CN1583794A - Targeted anti-tumour fused protein containing adenovirus E40rf4 protein - Google Patents

Abstract

The present invention includes recombinant human epidermal growth factor-adenovirus E4orf4 fusion protein, a fusion gene encoding the fusion protein, an expression vector containing the fusion gene, a method for secretory expression of the recombinant fusion protein using methylotrophic yeast, and A pharmaceutical composition containing the protein and its therapeutic use.

Description

Targeted anti-tumor fusion protein containing adenovirus E4orf4 protein

technical field

The present invention relates to recombinant human epidermal growth factor-adenovirus early transcription region 4 open reading frame protein product (E4orf4) fusion protein, a fusion gene encoding the fusion protein, an expression vector containing the fusion gene, and methyl nutrition A method for secretory expression of the recombinant fusion protein by type yeast, a pharmaceutical composition containing the protein and its therapeutic application.

Background technique

The human adenovirus gene early transcription region 4 (E4) transcription unit contains at least 7 open reading frames, and their respective products have no obvious similarity. The product encoded by the fourth open reading frame (E4open reading frame 4, E4orf4) is a 14kDa small molecule protein consisting of 114 amino acids, and its amino acid sequence has no homology with any known protein. E4orf4 protein has a highly conserved sequence, which is RXKRRXRRRR, and it also has a proline-rich sequence at the amino terminus, which may provide a potential Src homology region 3 (SH3) binding site. Deletion of any small fraction of amino acids from the polypeptide chain, including the amino- and carboxy-terminal regions, produces nonfunctional and often unstable mutants. In the early stage of adenovirus infection, the E4orf4 gene is transcribed by the E4 promoter to produce mRNA. Although transcription stops at the late E4 promoter, E4orf4 protein remains at a constant level in cells. This shows that E4orf4 protein is highly stable in cells (Branton PE and Roopchand DE. Oncogene, 2001; (20): 7855-7865; Marcellus RC, Chan H, Paquette D et al.J Virol, 2000; 74 (17): 7869-7877).

Many viruses induce apoptosis during their life cycle to kill infected cells. Human adenovirus infection can form a complex process of induction and inhibition of apoptosis. Adenovirus early region 1A (E1A) products lead to p53-dependent apoptosis by enhancing the activity and stability of p53 gene. Early region 1B (E1B) encodes two proteins of 55kDa and 19kDa, together with E4orf6 protein, inhibits apoptosis by blocking the toxic effect of E1A and produces a large amount of virus. Further experiments found that p53-deficient cells infected with adenovirus expressing complete E1A could still induce apoptosis, suggesting that this process was caused by other viral products expressed under the control of the E1A transactivation region. After a series of experiments, the cytotoxic function was finally located in the E4orf4 protein. In the absence of other adenovirus proteins, the E4orf4 protein has high cytotoxicity and induces apoptosis independent of p53. The pcDNA plasmid expressing E4orf4 can induce tumor cells The mortality rate of E4 is 80-90%, and it is the only E4 product that can kill cells alone. Adenovirus E4orf4 protein is a multifunctional viral regulatory protein. In vitro experiments, it can specifically induce the apoptosis of transformed cells, but has no effect on normal cells; the apoptosis induced by E4orf4 protein is p53-independent Yes, its apoptosis-inducing effect requires the interaction with protein phosphatase 2A; E4orf4 protein triggers the cytoplasmic expression of apoptosis by regulating the activity of Src kinase (Maecellus RC, Tendom JG, Wu T et al.J. Virol.1996, 70:6207-6215; Marcellus RC, Lavoie JN, Boivin D et al.J.Virol.1998, 72:7144-7153; Shtricheman R, Sharf R, Barr H et al.Proc.Natl.Acad. Sci. USA. 1999, 96: 10080-10085).

A large number of research reports have confirmed that EGF receptors are overexpressed in most tumor cells such as breast, lung, brain, bladder, kidney, and prostate, and their cell lines are often inhibited by EGF after growth in vitro or transplantation. It has been shown that chemically linking RNase 1 and recombinant EGF to form a complex EGF-RNase 1 has a dose-dependent cytotoxic effect on breast cancer and squamous cell carcinomas with high expression of EGF receptors, that is, the more expressed EGF receptors The higher the cell line, the more obvious the cytotoxic effect. Recently, Dmitriev et al. expressed the CAR-EGF fusion protein (CAR, Coxsackie virus and adenovirus receptor) using the baculovirus expression system, and effectively transported the adenovirus vector through the targeting effect of EGF and EGF receptor binding. In tumor cells, the release of reporter genes is increased (Yang Yuan, Huang Bingren, Cai Liangwan, etc. Chinese Academy of Medical Sciences Journal. 1996, 18(3): 171; Ueda M, PsarrasK, Jinno H et al. Breast Cancer 1997, 4( 4): 253-255; Dmitriev I, Kashentseva E, Rogers BE et al. J. Virol. 2000, 74(15): 6875-6884).

Due to the high proportion of p53 gene mutations in human tumors and the poor prognosis of these tumors with conventional treatment, it is very meaningful to try to find a new way to treat p53 mutant tumors. Adenovirus is a promising gene therapy vector, but in view of the fact that adenovirus gene therapy provides many benefits, the related technology is not yet fully mature, and there are certain risks in the safety of treatment. Therefore, it is still a research hotspot in the field of tumor treatment to further explore methods and drugs for treating with adenovirus.

Contents of the invention

The invention relates to a recombinant human epidermal growth factor-adenovirus E4orf4 fusion protein, a pharmaceutical composition containing the fusion protein and its use for targeted tumor therapy. In one embodiment, the fusion gene encoding the fusion protein of the present invention has the sequence of SEQ ID NO: 1, and the amino acid sequence of the fusion protein is shown in SEQ ID NO: 2.

Another aspect of the present invention relates to a fusion gene encoding the fusion protein, an expression vector and an engineering strain containing the fusion gene. According to a kind of genetic engineering bacterial strain of the present invention, named after Pichia pastoris GS115-3 EGF-E4orf4, on July 15th, 2002, it was preserved in the General Microorganism Center (CGMCC) of China Microorganism Culture Collection Management Committee, and its preservation No. 0771.

Description of drawings

Figure 1: Construction of α-factor leader peptide-EGF-E4orf4 gene fragment by overlapping PCR;

Figure 2: Electrophoretic pattern of restriction enzyme digestion identification of recombinant plasmid pUC-EGF-E4orf4;

Figure 3: Schematic diagram of site-directed mutation of recombinant plasmid pUC-EGF-E4orf4;

Figure 4: Physical map of plasmid pAO815, pAO815 consists of 7709 nucleotides, of which bases 1-940 are 5'AOX1 promoter fragments, bases 943-948 are EcoRI sites, and bases 950-1277 are 3'AOX1 Transcription terminator (TT), base 4199-1665 is HISORF, base 4554-5310 is 3'AOX1 fragment, base 6394-5740 is the start of ColE1, base 7399-6539 is ampicillin resistance gene;

Figure 5: Construction diagram of recombinant plasmid pAO-EGF-E4orf4;

Figure 6: The electrophoresis diagram of the enzyme digestion identification of the recombinant plasmid pAO-EGF-E4orf4;

Figure 7: Construction diagram of recombinant plasmid pAO-3EGF-E4orf4;

Figure 8: The electrophoresis diagram of the enzyme digestion identification of the recombinant plasmid pAO-3EGF-E4orf4;

Figure 9: Comparison of Mut ⁺ and Mut ^s transformants;

Figure 10: SDS-PAGE electrophoresis of EGF-E4orf4 secreted by GS115-3EGF-E4orf4;

Figure 11: Western Blot analysis of EGF-E4orf4 secreted by GS115-3EGF-E4orf4.

Figure 12: Results of recombinant EGF-E4orf4 fusion protein purified by cation exchange chromatography;

Figure 13: BT325 cell viability detected by MTT colorimetry;

Figure 14: MTT colorimetric method to detect MDA-MB-231 cell viability;

Figure 15: MDA-MB-231 cells cultured for 72 hours, flow cytometry analysis of cell apoptosis;

Figure 16: MDA-MB-231 cells were co-cultured with the fusion protein EGF-E4orf4 (10 μg/3×10 ⁵ cells) for 72 hours, and the apoptosis was analyzed by flow cytometry;

Figure 17: MDA-MB-231 cells were co-cultured with the fusion protein EGF-E4orf4 (15 μg/3×10 ⁵ cells) for 72 hours, and the cell apoptosis was analyzed by flow cytometry;

Figure 18: MDA-MB-231 cells were co-cultured with the fusion protein EGF-E4orf4 (20 μg/3×10 ⁵ cells) for 72 hours, and the apoptosis was analyzed by flow cytometry;

Figure 19: MDA-MB-231 cells were co-cultured with the fusion protein EGF-E4orf4 (25 μg/3×10 ⁵ cells) for 72 hours, and the apoptosis was analyzed by flow cytometry.

specific implementation plan

According to one aspect of the present invention, a recombinant human epidermal growth factor (EGF)-adenovirus E4orf4 fusion protein is provided, which comprises human epidermal growth factor, a linker, and adenovirus E4orf4 protein from the N-terminus to the C-terminus in sequence. In a preferred embodiment, the amino acid sequence of the fusion protein is shown in SEQ ID NO:2.

According to another aspect of the present invention, the present invention provides a fusion gene encoding the fusion protein of the present invention.

Preferably used in the fusion gene of the present invention is an artificially synthesized EGF gene fragment encoding 51 amino acids, which is described in Chinese Patent Application No. 97115284.5 for details. The E4orf4 protein gene sequence of the adenovirus in the fusion gene of the present invention is preferably a modified E4orf4 protein gene sequence encoding adenovirus A5, and the modified sequence is to mutate the 228th base from T to G through site-directed mutation, so that The restriction endonuclease BglII cutting site is eliminated, but the amino acid sequence is not changed, so as to facilitate the construction of yeast multi-copy tandem expression vector. The linker between the two gene fragments of EGF and E4orf4 is preferably a gene sequence encoding 11 amino acids composed of glycine and serine, so that it can form an arm to ensure that the EGF and E4orf4 proteins can not interfere with each other and form an independent Higher structure, exercising normal biological activity. In a preferred embodiment, the gene sequence encoding EGF-E4orf4 fusion protein of the present invention is shown in SEQ ID NO: 1.

According to still another aspect of the present invention, an expression vector comprising the fusion gene of the present invention and an engineering bacterium containing the expression vector are provided. Preferably, the expression vector of the present invention is an expression vector of an integrated single-copy or three-copy EGF-E4orf4 fusion protein capable of secretory expression, which is to combine the DNA fragment encoding the yeast α-factor leader peptide with the EGF-E4orf4 fusion protein by using overlapping PCR. The gene sequence of the E4orf4 fusion protein is fused, and the Kozak sequence is added before the α-factor leader peptide, and then cloned to the downstream of the alcohol oxidase promoter (AOX1) to construct. The single-copy and three-copy expression vectors of the present invention are, for example, pAO-EGF-E4orf4 and pAO-3EGF-E4orf4, as shown in FIG. 5 and FIG. 7 . Preferably, the engineering bacterium of the present invention is methylotrophic yeast (also known as Pichia pastoris) comprising the expression vector of the present invention, especially Pichia pastoris GS115-3 EGF-E4orf4, which was established in 2002 It was deposited in the General Microorganism Center (CGMCC) of the China Microbiological Culture Collection Management Committee on July 15, and its preservation number is 0771. The strain is an integrated Mut ⁺ genotype strain (GS115-3EGF-E4orf4) screened after transforming Pichia pastoris strain GS115 with 3 copies of the vector. After high-density culture and induced expression, the strain can secrete EGF-E4orf4 fusion protein. The molecular weight of the secreted EGF-E4orf4 fusion protein was consistent with the prediction. Using rabbit anti-human EGF polyclonal antibody and rabbit anti-adenovirus E4orf4 protein antiserum for Western Blot analysis, it was confirmed that the specifically expressed protein band and the two antibodies were positively reacted, and it was a fusion protein composed of EGF and E4orf4. . The expressed protein is separated and purified by column chromatography, so that the purification degree can reach 90% or higher.

In addition, in the present invention, two terminators TAA and TGA are connected to the end of the coding sequence of the 176th amino acid of the EGF-E4orf4 fusion protein. This design is beneficial to the separation and purification of gene expression products and the termination of gene transcription. The addition of Kozak sequence provides conditions for high-efficiency expression of EGF-E4orf4 fusion protein.

The fusion protein of the present invention has tumor-targeting properties, and can specifically kill tumor cells, especially tumors with p53 gene defects. The targeting property utilizes the high expression of EGF receptors on the surface of most tumor cells, and the fusion protein contains EGF , through the combination of EGF and EGF receptor, E4orf4 protein can be brought into tumor cells. E4orf4 is a protein that can produce high cytotoxicity alone in adenovirus, which can induce tumor cells to undergo p53-independent apoptosis, thereby achieving tumor growth. targeted therapy. The activity of the fusion protein of the present invention was verified as in the Example section.

Another aspect of the present invention relates to a pharmaceutical composition containing a pharmaceutically effective amount of EGF-E4orf4 fusion protein as an active ingredient. Such compositions contain pharmaceutically acceptable carriers, adjuvants, diluents or fillers. The pharmaceutical composition can be administered by injection, or locally administered through interventional treatment of tumors, in short, it can be used for the treatment of tumors according to clinical needs.

Hereinafter, the present invention will be further described with reference to the following examples.

Example 1: Construction of α-factor leader peptide-EGF-E4orf4 gene fragment by overlapping PCR

In this example, the following PCR primers were used:

Primer 1 (31-mer):

5'- GGAATTC ACC ATG AGA TTT CCT TCA ATT TTT-3'

EcoRI Kozak

Primer 2 (45-mer):

5’-ACC ACC ACC GGA ACC ACC ACC ACC TTC CCA CCA

CTT CAA GTC TCT-3’

Primer 3 (45-mer):

5’-GGT TCC GGT GGT GGT GGT TCC TCC ATG GTT CTT

CCA GCT CTT CCC-3’

Primer 4 (31-mer):

5'- GGAATTC TCA TTA CTG TAC GGA GTG CGC CGA-3'

EcoRI kill code

Primer 1 contains an EcoRI restriction site and Kozak sequence, and is complementary to the 5' end of the α-factor leader peptide; primer 2 is complementary to the 3' end of EGF, and partly coded for the amino acid arm consisting of glycine and serine. sequence; primer 3 is complementary to the 5' end of E4orf4, and a part of the sequence encoding the amino acid arm composed of glycine and serine is added, and this sequence is complementary to primer 3; primer 4 is complementary to the 3' end of E4orf4, and two A terminator and an EcoRI restriction site.

Using the plasmid pYA41EGF as a template (Huang Bingren, Zhang Dai, Chi Laishun, et al. Chinese Academy of Medical Sciences Journal 1989, 11(5): 331-337), amplify with primer 1 and primer 2 to obtain a DNA fragment containing the EcoRI recognition site , Kozak sequence, α-factor leader peptide, EGF and 8 amino acid arms consisting of glycine and serine. Using the plasmid pUC-E4orf4 as a template, the DNA fragment amplified by primers 3 and 4 contained eight amino acid arms consisting of glycine and serine, E4orf4 and EcoRI recognition sites. Further, using two DNA fragments as templates, annealing is performed first to make the bases of the 5 amino acids of the amino acid arms at both ends complementary, and then primers 1 and 4 are used to amplify, that is, the two ends are EcoRI recognition sites, containing complete Alpha-factor leader peptide-EGF-E4orf4 gene fragment. The gene fragment was digested with EcoRI and then ligated into the pUC18 plasmid vector, and the resulting recombinant clone was pUC-EGF-E4orf4, as shown in Figure 1. Sequencing results confirmed that it was completely consistent with the design.

Example 2: Enzyme digestion identification of recombinant plasmid pUC-EGF-E4orf4

Identification method: 2 μg of the recombinant plasmid pUC-EGF-E4orf4 was digested with EcoRI enzyme at 37°C for 2 hours, electrophoresed on a 1.0% agarose gel, stained with ethidium bromide, and a total of 261 fragments of the pUC18 vector and the leader peptide encoding α-factor-EGF-E4orf4 were seen. An about 800bp DNA clone fragment of amino acids, 2 terminators, Kozak sequence and EcoRI site.

The enzyme digestion identification of the plasmid is shown in Figure 2, in which: Swimming lane 1: λDNA/HindIII, the fragments from large to small are: 23130bp, 9414bp, 6557bp, 4361bp, 2322bp, 2021bp, 564bp. Lane 2: recombinant plasmid pUC-EGF-E4orf4. Lane 3: pUC-EGF-E4orf4/EcoRI 2.69kb (vector), 800bp (insert fragment). Lane 4: DNA Marker DL2000, fragments from large to small are: 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp.

Example 3: Site-directed mutagenesis of recombinant plasmid pUC-EGF-E4orf4

Since the construction of a multi-copy expression vector requires the use of the homologous enzymes BglII and BamHI for the tandem connection of the expression units, the exogenous gene fragments cannot contain the cutting points of BglII and BamHI. The E4orf4 gene fragment contains a BglII recognition site, which needs to be removed by site-directed mutagenesis without changing the amino acid sequence.

Stratagene's kit (QuickChangeTM Site-Directed Mutagenesis Kit) was used for site-directed mutagenesis, and the strategy is shown in Figure 3. The primer sequences for site-directed mutagenesis are as follows:

Primer 1:

5’-CGG AGA CGC AGA TCG GTT TGT CAC GCC CGC-3’

Primer 2:

5’-GCG GGC GTG ACA AAC CGA TCT GCG TCT CCG-3’

Using the plasmid pUC-EGF-E4orf4 obtained in Example 1 as a template, PCR amplification of site-directed mutagenesis was performed, and then transformed into Escherichia coli DH5α. DNA sequence analysis of the transformants confirmed that the 228th base of the E4orf4 gene changed from T to G. The mutated pUC-EGF-E4orf4 plasmid was digested with EcoRI to obtain the gene fragment encoding α-factor leader peptide-EGF-E4orf4, which was ligated with the yeast expression vector pAO815 (purchased from Invitrogen) linearized by EcoRI to obtain the recombinant plasmid pAO -EGF-E4orf4 (Figure 5).

Example 4: Enzyme digestion identification of recombinant plasmid pAO-EGF-E4orf4

The 944 bp downstream of the AOX1 promoter of the pAO815 vector is the EcoRI cloning site. After the recombinant plasmid pAO-EGF-E4orf4 is digested with EcoRI, an insert fragment of about 800 bp can be seen. Recombinant plasmid pAO-EGF-E4orf4 needs to be identified by enzyme digestion to screen out positive clones. There is a PstI cutting point at the 24th base of the α-factor leader peptide, and there is also a PstI cutting point at the 6858th base of the pAO815 vector. Point, after the recombinant plasmid pAO-EGF-E4orf4 is digested with PstI, two fragments of 1.8kb and 6.7kb can be obtained by forward cloning, and two fragments of 2.6kb and 5.9kb can be obtained by reverse cloning.

The electrophoresis results of enzyme digestion and identification of the plasmid are shown in Figure 6. In the figure: Swimming lane 1: DNA molecular weight marker DL2000, the fragments from large to small are: 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp. Lane 2: pAO-EGF-E4orf4/EcoRI, 7.7 kb (vector), 800 bp (insert). Lane 3: pAO-EGF-E4orf4/PstI, 6.7kb, 1.8kb. Lane 4: pAO-EGF-E4orf4. Lane 5: λDNA/HindIII, the fragments from large to small are: 23130bp, 9414bp, 6557bp, 4361bp, 2322bp, 2021bp, 564bp.

Example 5: Construction of three-copy expression vector pAO-3EGF-E4orf4

The pAO-EGF-E4orf4 obtained in Example 3 was cut out with BglII and BamHI to a fragment containing the AOX1 promoter, the α-factor leader peptide-EGF-E4orf4 coding sequence and the AOX1 terminator, and then used BglII and BamHI to make it self-ligated in vitro. BamHI enzyme digestion, at this time, only the dimer formed by the connection of BglII and BamHI in the reaction system, that is, the fragments whose two ends are respectively BglII and BamHI cannot be cut again. Connect this reaction product to the BamHI site of alkaline phosphatase-treated pAO-EGF-E4orf4, transform DH5α recipient bacteria, and obtain three copies of EGF-E4orf4 expression units in the same direction after screening and identification of recombinant DNA Recombinant pAO-3EGF-E4orf4. See Figure 7 for the plasmid construction map, and Figure 8 for the plasmid digestion identification map.

pAO815 will be digested with BglII and BamHI to produce three fragments from large to small: 4028bp, 2405bp, and 1279bp; The last fragment comes from the insertion of the 800bp fragment into the 1279 fragment. After digesting pAO-3EGF-E4orf4 with BglII and BamHI, three fragments from large to small, 6237bp, 4028bp, and 2405bp, will be produced. The 6237bp fragment is a triploid of the 2079bp fragment.

In Fig. 8: Swimming lane 1: λDNA/HindIII, the fragments from large to small are: 23130bp, 9414bp, 6557bp, 4361bp, 2322bp, 2021bp, 564bp. Lane 2: pAO815/BglII+BamHI, the fragments from large to small are: 4028bp, 2405bp, 1279bp. Lane 3: pAO-EGF-E4orf4/BglII+BamHI, the fragments from large to small are: 4028bp, 2405bp, 2079bp. Lane 4: pAO-3EGF-E4orf4/BglII+BamHI, the fragments from large to small are: 6237bp, 4028bp, 2405bp.

Embodiment 6: the preparation of host bacterium, transformation and the screening of engineered bacterium

Methylotrophic yeast (ie, Pichia pastoris, Pichia pastoris) was used as the expression host. Methylotrophic yeast has many advantages: (1) It contains a unique AOX1 (alcohol oxidase) promoter, and the expression can be strictly regulated by methanol. (2) It has a complete fermentation method, can be cultured continuously at high density, and has high protein expression yield. (3) Strong viability, easy to handle, and can grow in cheap non-selective medium. To this end, the multi-copy EGF-E4orf4 expression vector pAO-3EGF-E4orf4 obtained in Example 5 used in the present invention, its transformation, screening and expression processes are as follows.

The materials used in Examples 6 to 9 are:

10X YNB: Dissolve 134g of yeast base containing ammonium sulfate but no amino acid in 1000ml of water, filter aseptically and set aside.

500X biotin (0.02% biotin): 20mg VitH dissolved in 100ml water, sterile filtered, stored at 4°C for later use.

10X D (20% glucose): 200g glucose dissolved in 1000ml water, sterile filtered, stored at 4°C for later use.

10X M (5% Methanol): Dissolve 5ml methanol in 95ml water, sterile filter, store at 4°C for later use.

10X GY (10% glycerol): Dissolve 100ml glycerin in 900ml water, autoclave, store at room temperature for later use.

1M Phosphate Buffer, pH 6.0: Mix 132ml _1M K2HPO4, 868ml _1MKH2PO4 .

BMG and BMMY: 100mM phosphate buffer pH6.0, 1.34% YNB, 1% glycerol or 0.5% methanol, 0.00004% biotin, BMMY also contains 1% yeast extract, 2% peptone.

YPD medium: 1% yeast extract, 2% peptone, 2% glucose.

MD and MM: 1.34% YNB, 0.00004% biotin, 2% glucose or 0.5% methanol.

The specific steps of preparation and linearization of the expression vector 3EGF-E4orf4:

Preparation of moderate amount of plasmid DNA: Inoculate the strain containing the expression vector in 50ml of LB medium containing 100μg ampicillin/ml, culture overnight on a shaker at 37°C, collect the bacteria by centrifugation, and extract with the plasmid purification kit from QIAGEN Purify plasmid DNA.

Take 10 μg of pAO-3EGF-E4orf4 and digest it completely with BglII, extract with phenol-chloroform-isoamyl alcohol, precipitate with ethanol, and freeze-dry for later use.

Example 7: Transformation of host bacteria with pAO-3EGF-E4orf4

Pichia pastoris GS115(his4-) was purchased from Invitrogen, inoculated on YPD plates, cultured at 30°C for 2 days, and isolated from single clones. The single clone was re-inoculated in 5ml YPD liquid medium, cultured at 30°C until the _OD600 was about 1.3-1.5, collected by centrifugation, rinsed with sterile cooling water, and suspended in 1ml of ice-cold 1M sorbitol solution after centrifugation. Take 80 μl of host bacteria and 5 μl of 10 μg linearized pAO-3EGF-E4orf4 prepared in Example 6 and mix them in an electroporation cup, and pre-cool at 0° C. for 5 minutes. The Bio-Rad gene puncher (Gene-pluserII) program was set to generate an electric pulse of 7500V/cm, 10ms to complete the transformation. Immediately add 1ml of ice-cold 1M sorbitol to the electroporation cup, take 200μl to spread the MD culture plate, and culture at 30°C until the recombinants appear.

Embodiment 8: screening Mut ⁺ and Mut ^s transformants

Methylotrophic yeast contains two AOX genes, AOX1 and AOX2. The expression vector is linearized after cutting with different enzymes (such as BglII, NotI or SalI, StuI, etc.), so that it is integrated at the position of the AOX1 or HIS4 gene in the yeast genome. When the AOX1 gene of yeast is replaced and lost, Mut ^s phenotype (methanolutilization slow) will be produced, they will use the weak AOX2 gene to start the synthesis of AOX, and grow slowly in the medium containing methanol. The wild-type yeast grows normally in the methanol-containing medium, which is called the Mut ⁺ phenotype.

Prepare MD-agarose plates and MM-agarose plates. In the case of using the two strains GS115-Albumin (His ⁺ Mut ^s ) and GS115-Gal (His ⁺ Mut ⁺ ) as controls, use a sterile toothpick to symmetrically transfer the transformants obtained in Example 7 from the MM plate to the MD plate Inoculate on two kinds of plates, culture at 30°C, and observe the growth of transformants. The Mut ^s phenotype grows slowly in the medium containing methanol, and the Mut ⁺ phenotype grows normally in the medium containing methanol, see Figure 9. The His ⁺ Mut ⁺ transformant was picked as a genetic engineering strain, named GS115-3EGF-E4orf4, and the expression of EGF-E4orf4 was observed.

Example 9: Expression of EGF-E4orf4

The genetically engineered strain GS115-3EGF-E4orf4 obtained in Example 8 was cultured using shake flasks. Inoculate in 25ml of BMG medium as a single clone or strain stored at -80°C, culture at 30°C for 18-24h until OD ₆₀₀ = 10-12, collect the bacteria by centrifugation, transfer to 250ml of BMMY medium, and culture at 30°C, The expression was induced by adding methanol at a concentration of 1% every 24 hours, and the optimal induction time was determined to be 96-120 hours. The culture supernatant was collected by centrifugation for the detection of EGF-E4orf4 fusion protein.

Embodiment 10: Detection of recombinant EGF-E4orf4 fusion protein

The recombinant EGF-E4orf4 fusion protein obtained in Example 9 above was analyzed by SDS-PAGE protein electrophoresis. Use Bio-Rad protein electrophoresis device, pour 15% separation gel, collect the culture supernatant, add protein electrophoresis sample buffer, denature at 100°C for 5 minutes, load electrophoresis, 100V constant voltage, electrophoresis until bromophenol blue gel, stop electrophoresis , stained with silver nitrate.

The results are shown in Figure 10. Referring to the protein molecular weight standard, the molecular weight of the EGF-E4orf4 fusion protein is about 20kD, which conforms to the molecular weight of the EGF-E4orf4 fusion protein with amino acid composition. In Figure 10: Swimming lane 1: GS115-3 EGF-E4orf4 expression supernatant, the arrow shows the EGF-E4orf4 fusion protein band. Swimming lane 2: Protein low molecular weight standards, the fragments from large to small are: 43kD, 29kD, 18.4kD, 14.3kD, 6.2kD.

Western Blot analysis was also performed. After the recombinant EGF-E4orf4 fusion protein was separated by SDS-PAGE electrophoresis, it was transferred to a nitrocellulose membrane, and rabbit anti-human EGF polyclonal antibody (purchased from Santa Cruz Company) or rabbit anti-E4orf4 protein antiserum ( Prepared by the present inventors according to a conventional method) and incubated with nitrocellulose membrane at room temperature for 1 hour, after rinsing, HRP-labeled goat anti-rabbit IgG antibody was added for 1 hour at room temperature, and detected by ECL chemiluminescence colorimetry after rinsing.

The results are shown in Fig. 11, it can be seen that the EGF-E4orf4 fusion protein has a positive reaction with the human EGF antibody. In Fig. 11 : Swimming lane 1: protein low molecular weight standard, the fragments from large to small are: 43kD, 29kD, 18.4kD, 14.3kD, 6.2kD. Lane 2-3: GS115-3EGF-E4orf4 expression supernatant, the primary antibody is rabbit anti-human EGF polyclonal antibody. Swimming lane 4: GS115-3EGF-E4orf4 expression supernatant, the primary antibody is rabbit anti-E4orf4 antiserum, and the arrow shows the EGF-E4orf4 fusion protein band.

Example 11. Separation and purification of recombinant EGF-E4orf4 fusion protein

The yeast expression supernatant containing the recombinant EGF-E4orf4 fusion protein obtained in Example 9 was purified by cation exchange chromatography using the FPLC system of Pharmacia Company. Chromatography column: XK26, chromatography medium: SP Sepharose Fast Flow (strong cation), column volume 50ml. The yeast expression supernatant was diluted with 6-7 times the volume of buffer A (50mMNaAC/HAC, pH4.0), and filtered through a 0.45μm microporous membrane; after equilibrating the medium with 5 column volumes of buffer A, load the sample, The flow rate is 5ml/min. After the loading peak is washed to the baseline with buffer A, the flow rate is adjusted to 3ml/min, and buffer B with different salt concentrations (B1: 0.5M NaCl, 50mM NaAC/HAC, pH4.0; B2 : 1.0M NaCL, 50mM NaAC/HAC, pH4.0; B3: 1.5M NaCL, 50mM NaAC/HAC, pH4.0) for step-by-step elution, separate tubes to collect elution peaks, and SDS-PAGE protein electrophoresis identification. With 0.5M and 1.0M NaCl, 50mM acetate buffer, pH 4.0, most of the impurity proteins can be eluted, and then eluted with 1.5M NaCl, 50mM acetate buffer, pH 4.0, a better purification effect can be obtained . The eluate containing the fusion protein identified by electrophoresis was collected, desalted by dialysis, concentrated with PEG-10000, sterilized by filtration with a 0.22 μm filter membrane, stored at -20°C, and quantified for protein. See Figure 12.

In Fig. 12: Lane 1: Yeast expression supernatant of fusion protein EGF-E4orf4. Swimming lane 2: Protein low molecular weight standards, fragments from large to small are: 43kD, 29kD, 18.4kD, 14.3kD. Swimming lane 3: the eluate collected by cation exchange chromatography, the arrow indicates the recombinant EGF-E4orf4 fusion protein.

Embodiment 12.MTT colorimetric assay measures the influence of recombinant EGF-E4orf4 fusion protein on cell viability

Mitochondrial dehydrogenase in living cells can convert the dye MTT (dimethyl thiazolium blue diphenyl tetrazolium bromide) into insoluble purple substance (formazan) particles, the color of the latter after being dissolved by the organic solvent DMSO (expressed by OD value), which can reflect the metabolic level of living cells. Dead cells do not have this enzyme activity. Therefore, within a certain range of cell numbers, the formation of MTT crystals is directly proportional to the number of living cells. In this example, this assay was used to test the effect of the fusion protein on cell viability.

Specifically, monolayer cultured cells were digested with 0.25% trypsin, blown into a single cell suspension with a medium containing 10% fetal bovine serum, and seeded in a 96-well culture plate with 5000 cells per well, with a volume of 200 μl per well. , 37°C, 5% _CO2 culture, 20-24 hours after the cells are completely adhered to the wall, discard the supernatant, replace with the medium containing 5% fetal bovine serum, 200μl per well, 37°C, 5% _CO2 culture . The fusion protein EGF-E4orf4 obtained in Example 11 was serially diluted from a concentration of 1 ng-15 μg/ml with PBS, and the EGF standard was used as a control, diluted according to the concentration corresponding to the molecular ratio of the fusion protein EGF-E4orf4, and each well was sequentially diluted Add the diluted continuous gradient fusion protein EGF-E4orf4 solution and the EGF solution corresponding to the equimolecular ratio, set 3 parallel wells for each concentration, repeat 2 plates, and incubate with the cells at 37°C, 5% CO ₂ for 2-3 sky. Add 20 μl of MTT solution (5 mg/ml) to each well, continue to incubate for 4-5 hours at 37°C, 5% CO ₂ , carefully suck off the culture supernatant in the well, add 100 μl DMSO to each well, and shake for 10 minutes to make the crystals Fully dissolve. The light absorption value of each well at a wavelength of 570 nm was measured on an enzyme-linked immunosorbent detector. Calculate the mean value of OD value and input it into SPSS analysis software, and the result is expressed in an automatically generated graph, the abscissa shows the concentration of the stimulus, which is the final concentration (ng/ml) of the fusion protein E4orf4, and the ordinate is the OD value.

It can be seen in Figure 13: BT325 cells (human brain glioma cells, established by Beijing Tiantan Hospital) were co-cultured with the fusion protein EGF-E4orf4 for 44 hours, at a higher concentration from 2.5 μg/ml to 12.5 μg/ml, The cell viability continued to decline. When the fusion protein reached 12.5 μg/ml, which is the highest concentration of the fusion protein used in this experiment, the cell viability dropped to the minimum, and the OD ₅₇₀ was 0.183, which was far lower than that of the negative control group cells without stimulants. Vitality, its OD ₅₇₀ is 0.276, which is completely different from the cells in the positive control group added with EGF. At this time, the viability of the cells in the EGF group with the corresponding molecular concentration is still maintained at a high level, and two separate viability curves appear. Phenomenon. This result proves that the fusion protein EGF-E4orf4 can enter BT325 cells mediated by EGF, and when its concentration accumulates to a high level, it can produce obvious cytotoxic effect on BT325 cells.

Visible in Fig. 14: MDA-MB-231 cell (human breast cancer cell, ATCCNumber: HTB-26) and fusion protein EGF-E4orf4 are co-cultured for 68 hours, from concentration increasing to 500ng/ml, cell viability drops to the minimum, low In the control cell group without stimulator, at the later concentration of stimulant (2.5μg/ml-15μg/ml), the cell viability was always lower than or close to that of the control cell group without stimulator, while the EGF control group The cell viability was always higher than that of the no-stimulator group, indicating that the fusion protein EGF-E4orf4 could inhibit the viability of MDA-MB-231 cells.

Example 13. Detection of cell apoptosis by flow cytometry

During the process of cell apoptosis, due to the condensation of cytoplasm and chromatin, the cleavage of cell nucleus produces apoptotic bodies, which lead to changes in the light scattering properties of cells. Compared to live or necrotic cells, apoptotic cells have a specific type of light scatter - low forward scatter and high side scatter. Flow cytometry can measure changes in light scattering of cells, so it is widely used in the observation, detection and analysis of cell apoptosis. According to the results of the MTT colorimetric experiment, the concentration required for the fusion protein when the viability of MDA-MB-231 cells drops to the lowest point is the starting point, and the concentration of the fusion protein that can basically saturate the cell surface EGFR is the end point, within this range Set a series of continuous fusion protein concentrations (10 μg/3×10 ⁵ cells-25 μg/3×10 ⁵ cells), co-culture with MDA-MB-231 cells, L15 medium containing 5% FBS, after 72 hours of culture Cell changes were detected by flow cytometry. The results are shown in Figures 15-19. It can be seen that the fusion protein EGF-E4orf4 can induce apoptosis of MDA-MB-231 cells. Within a certain concentration range, the degree of cell apoptosis is positively correlated with the concentration of the fusion protein. As the fusion protein grows from scratch, the apoptosis also grows from scratch; the fusion protein increases from 10 μg to 25 μg, and the cells undergo apoptosis from a small amount to obvious apoptosis until the cells enter the late stage of apoptosis. It can be concluded that the fusion protein EGF-E4orf4 can induce apoptosis of MDA-MB-231 cells, and MDA-MB-231 cells are relatively sensitive to the cytotoxicity of the fusion protein, and the cells at a lower concentration of the fusion protein Apoptosis is evident.

As shown in Figure 15: MDA-MB-231 cells were cultured without fusion protein for 72 hours, and 92.1% of the cells grew well.

As shown in Figure 16: MDA-MB-231 cells were co-cultured with the fusion protein EGF-E4orf4 (10 μg/3×10 ⁵ cells), and a small number of cells had undergone apoptosis, accounting for about 15.4%.

As shown in Figure 17: MDA-MB-231 cells were co-cultured with the fusion protein (15 μg/3×10 ⁵ cells), 28.2% of the cells escaped from the cell cycle, and the cells underwent obvious apoptosis. Moreover, it can also be seen from the figure that the characteristics of early apoptosis of cells, that is, a slope or sub-peak produced by apoptotic cells appears before the G ₀ /G ₁ peak. This is because in the early and middle stages of apoptosis, nuclear DNA breaks and is degraded into DNA fragments that are integer times the size of nucleosomes. Arrows indicate hypodiploid peaks.

As shown in Figure 18: MDA-MB-231 cells were co-cultured with the fusion protein (20 μg/3×10 ⁵ cells), and 19.1% of the cells were apoptotic.

As shown in Figure 19: when the concentration of the fusion protein reaches 25 μg/3×10 ⁵ cells, 27.0% of the MDA-MB-231 cells undergo apoptosis. At this time, cell apoptosis has entered the late stage, and the nuclear DNA is degraded into nucleosome-sized DNA fragments, resulting in a peak at a very low PI light absorption value, as indicated by the arrow.

Sequence Listing

<110> Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences

<120> Targeted anti-tumor fusion protein containing adenovirus E4orf4 protein

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Claims (14)

1. A recombinant human epidermal growth factor (EGF)-adenovirus E4orf4 fusion protein, which consists of human epidermal growth factor, linker, and adenovirus E4orf4 protein from N-terminus to C-terminus. 2. The fusion protein according to claim 1, wherein the amino acid sequence of the fusion protein is as shown in SEQ ID NO: 2, which consists of human epidermal growth factor of 51 amino acid residues, a linker of 11 amino acid residues and E4orf4 protein composition of adenovirus A5. 3. A fusion gene having a nucleotide sequence encoding the fusion protein of claim 1 or 2. 4. The fusion gene as claimed in claim 3, which has the sequence of SEQ ID NO:1. 5. An expression vector containing at least one copy of the fusion gene of claim 3 or 4. 6. The expression vector according to claim 5, which is the plasmid pAO-EGF-E4orf4 shown in Figure 5 . 7. The expression vector according to claim 5, which is the plasmid pAO-3EGF-E4orf4 shown in FIG. 7 . 8. A host cell comprising the fusion gene of claim 3 or 4 or the expression vector of any one of claims 5-7. 9. The host cell of claim 8, which is a methylotrophic yeast cell. 10. The host cell according to claim 9, which is Pichia pastoris GS115-3EGF-E4orf4, and the accession number is CGMCC0771. 11. A method for producing the fusion protein according to claim 1 or 2, comprising culturing the host cell according to any one of claims 8-10 under conditions suitable for expression, and isolating and purifying the expressed fusion protein. 12. A pharmaceutical composition comprising the fusion protein of claim 1 or 2 and a pharmaceutically acceptable carrier. 13. Use of the fusion protein according to claim 1 or 2 in the preparation of a drug for treating malignant tumors. 14. The use according to claim 13, wherein said malignant tumor is a p53 gene-deleted tumor.

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