JPH0251597B2 - - Google Patents

Description

[Detailed description of the invention]

[Field of Technology] The present invention relates to plasmid pAQ440 and a DNA transport vector containing this as an active ingredient.
More specifically, the part containing the gene that specifies the biosynthesis of the jellyfish photoprotein aquarin and pBR322
~Novel plasmid created from SV40
Regarding pQA440. [Background of the Invention] Aquarin is a photoprotein that grows on the west coast of the United States, and when one molecule of aquarin specifically binds to two molecules of Ca ²⁺ , it reduces the emitter coelenterazine and emits light. On the other hand, since Ca ²⁺ is essential for the luminescence of aquarin, Ca ²⁺ can be specifically quantified using aquarin.
It is possible to measure Ca ²⁺ concentrations as small as 10 ^-7 M. Currently, there are many Ca ²⁺ -dependent enzymes in living organisms, and Ca ²⁺ deficiency can cause serious diseases, so this aquarium may be widely used in clinical tests, and it is also possible to inject it into cells. This allows applications such as intracellular Ca ²⁺ concentration measurement and cold light sources. However, this jellyfish grows only in the northwestern part of the west coast of the United States, only occurs in the summer, and the yield is only about 1 mg per 10,000 jellyfish, so its productivity is not sufficient and the supply is limited. cannot be guaranteed. Therefore, the present inventor conducted intensive research in order to solve the above-mentioned problem, and used transgenic engineering technology to develop the aquarin gene that essentially specifies the production of aquarin protein.
- It was isolated and prepared as a cDNA clone from mRNA using the Berg method. That is, we used recombinant DNA procedures to create a cDNA clone of the jellyfish photoprotein aquarin,
This cDNA clone was named plasmid pAQ440. [Configuration of the Invention] The nucleotide sequence contained in the plasmid of the present invention is as shown in FIG. Furthermore, the present invention
Includes DNA delivery vectors having as active ingredients the plasmid pAQ440 described above and/or functional equivalents of the nucleotide sequences described above. Functional equivalents herein refer to nucleotide sequences that can be used in substantially the same manner to obtain substantially the same results in the production of the jellyfish photoprotein aquarin by a suitable host. To further explain the invention, FIG. 2 shows a restriction enzyme map of the cloned plasmid pAQ440. In the figure, 〓〓 indicates the part containing the aquarin gene, 〓〓 indicates the Amp ^r part of pBR322,
-- portion indicates the Okayama-Berg vector portion. Table 1 shows the cleavage sites and the number of cleavage sites using restriction enzymes.

[Table] Figure 3 shows a detailed restriction enzyme map of the aquarin gene cloned into plasmid pAQ440 and the direction of nucleotide sequence determination by the Maxam-Gilbert method. In the figure, plasmid pAQ440, a cDNA clone, contains the entire gene of the jellyfish photoprotein aquarin and several hundred nucleotides of the 5' and 3' ranking sequences. The nucleotide sequence of the aquarin gene revealed in Figure 1 above is 958 bp (base pair),
Contains a 588bp open reading frame (translated portion). The method for preparing pAQ440, which is a cDNA clone of the present invention, will be explained in detail based on FIGS. 4 and 5. Extract total RNA from the rim of a jellyfish (Aequorea victoria) using the guanidine hydrochloride method ^* . Next, poly A ⁺ mRNA was purified by oligo-dT cellulose column chromatography, and Okayama
−A cDNA library was created using the Berg method ^* (Note*H.Okayama et al Mol.Cell Biol 2161
(1982)) (Figure 4 1). On the other hand, N of aquarin protein extracted from jellyfish
Determine the amino acid sequence near the end and the amino acid sequence after bromocyanolysis, and extract the corresponding DNA.
Chemically synthesize 14 nucleotides with the base sequence,
It was used as a probe for screening the cDNA library described above. The nucleotide sequence was determined, and the aquarin gene was identified from the amino acid sequence and amino acid composition of the aquarin protein (see Figure 4, 2). Used in Figure 5 1 related to the above explanation
Okayama-Berg vector and Oligo (dG)
Linker was prepared by the method shown in 1 and 2 of FIG. According to the present invention, when producing jellyfish photoprotein aquarin using a suitable host, the above-mentioned method is used to produce
This can be easily achieved by using a cloned aquarin cDNA gene. By cloning this gene, the amino acid sequence (primary structure of the protein) of the aquarin protein was revealed in the present invention. That is, by specifying the aquarin gene, it is possible to specify the amino acid sequence of the aquarin protein. This is obvious in the field of molecular biology. Furthermore, it is obvious that a gene that specifies one specific amino acid is determined by multiple amino acid codons (three nucleotides), which is functionally equivalent to the primary structure of aquarin protein revealed in the present invention. (aquarin protein with luminescent ability) is the same as that created using the same amino acid codon. [Effects of the Invention] As described above, it is clear that the photoprotein aquarin produced by the aquarin cDNA clone pAQ440 of the present invention is useful as a clinical medicine or reagent for quantifying trace amounts of Ca ²⁺ . A prokaryotic cell host (eg, E. coli) or a eukaryotic cell host (eg, a cultured cell system) can be used as a host for this clone in order to carry out the synthesis of aquarin. Such hosts are well known to those skilled in the art. Furthermore, the nucleotide sequence of the aquarin gene contained in the cDNA clone pAQ440 according to the present invention can be used for cloning into other vectors or hosts by methods well known to those skilled in the art. [Examples] The present invention will be explained below using Examples. Example 1 Creation of cDNA library: This will be explained according to FIG. (Step 1: Synthesis of cDNA) 116 g of the jellyfish cap rim was treated by the guanidine hydrochloride method to obtain 5 mg of total RNA. this thing
5.1 μg of poly A ⁺ messenger RNA (mRNA) was obtained by chromatography on a 50 μg oligo dT cellulose column. Of this, 3.8μg of poly A ⁺ RNA was lyophilized and 10μg of 5mM Tris-HCl (PH8.3) was added.
It was dissolved in buffer and incubated at 90°C for 1 minute. Next, pre-incubate at 37℃ for 5 minutes, add 10μ of reaction solution (described below), add 1.0U of reverse transcriptase (manufactured by Life Science), and heat for 60 minutes at 37℃.
Incubate for minutes. The composition of the reaction solution mentioned above was 500mM Tris-
HCl (PH8.3) buffer, 8.0mM MgCl ₂ , 300m
M KCl; 2μ, 20mM dNTP; 2μ, 1.4μ
g 1μOkayama-Berg vector DNA; 1μ
, ;H ₂ O; 2μ, 3000Ci/mmolα−[ ³² P]−
dCTP; 1μ 6mM dithiothreitol; 1μ
It is. After the reaction described above, add 2μ of 250mMEDTA, 1μ
The reaction was stopped by adding 10% SDS, extracted with the same amount of phenol-chloroform as the reaction system, 4M ammonium acetate of the supernatant of the extract was added, ethanol precipitated, and the precipitate was dissolved in 75% ethanol. After washing, it was dried in a vacuum desiccator. (Step 2: dC chain addition) Add 1.4μ of the reaction solution (described later) to the precipitate obtained in Step 1, dissolve it, and then add 1μ (15U).
terminal transferase (PL
(manufactured by Biochemical Inc.) and allowed to react at 37°C for 3 minutes, EDTA/SDS was added to stop the reaction, and DNA was recovered by a conventional method of extraction with phenol/chloroform. The composition of the reaction solution mentioned above was 1.4M cacodylic acid-0.3M Tris・KOH (PH
6.8) Buffer; 1.5μ, 10mM CoCl ₂ ;
1.5μ, 1mM dCTP; 1μ, α-[ ³² P]
dCTP (3000Ci1mmol); 1μ, _H2O ; 5.5μ,
1mM diothreitol; 1.5μ. (Step 3: Hind digestion) Add 1 μ of reaction solution (described later) to the recovered DNA obtained in Step 2, 8 μ of water, and Hind (20 U/μ).
After adding 1μ and reacting at 37°C for 60 minutes, DNA was collected by a conventional method. The composition of the reaction solution mentioned above is
200mM Tris-HCl (PH7.5). 70mM
_MgCl2 , 600mM NaCl. (Step 4: Transformation of RNA strand into DNA strand) cDNA digested with Hind in Step 3
1μ of primer DNA solution to 10μ of reaction solution (described later)
After incubating at 65°C for 2 minutes and 42°C for 30 minutes, cool on ice and remove the linker DNA containing the dG strand.
and cDNA primer DNA were annealed. The composition of the above reaction solution was 10mM Tris-HCl.
(PH7.5), 1mM EDTA, 0.1M, NaCl, 7ng
0:90 (dG) talied linker. After the above-mentioned annealing, it was left to stand overnight at 12°C under the following conditions. Reaction buffer 200mMTris-HCl PH7.540m
M MgCl ₂ 100mA (NH ₄ ) ₂ SO ₄ 10μ 5mMβ-NAD 2μ Water 7.6μ 400μg/ml E Coli ligase 0.7μ Furthermore, the following were added in sequence and reacted at 12℃ for 1 hour, then at 25℃ for another 3 hours Incubated. 4mM dNTP 1μ 5mM β-NAD 1μ 350U/μ polymerase 0.6μ 460U/μ RNase H 1μ (Step 5: Transformation) Transform the circular DNA obtained in Step 4 into competent cells DH1 prepared by a conventional method ^* . We conducted a transformation and obtained approximately 15,000 transformants. (Note *D. Hanakan, J. Mol. Biol. 166 557
(1983)) Through steps 1 to 5 above, jellyfish
A cDNA library was obtained. Screening method for aquarin gene: The amino acid sequence of aquarin protein isolated from jellyfish was determined. The N-terminus of the protein is Val Lys ( ) ( ) Asp Phe Asp Asm
Pro... After promo cyan processing, Met Asp Pro Ala Cys Gin Lys Leu Tyr
Gly…… Met(Phe)Asn(Phe)Leu Asp Val Asn
( ) Asn Gly... Next, four types of DNA corresponding to the above amino acids
was created using a synthesizer.

[Table] The ends of the above four types of DNA were prepared under the following conditions.
The 5' end was labeled with ^32P and used as a probe. 1μg: DNA 10μ: (0.5M TrisHCl, PH7.6, 0.1M
MgCl ₂ 50mM DTT, 1mM Spermindine
1mM EDTA) 50μ: γ- ³² P-ATP (3000Ci/mmol)
500μCi 40μ: Water 2μ: _T4 polynueclotide Kinase (TakaraF
-1) Using this probe, a cDNA library was screened by ^conventional colony hybridization to obtain a (positive) clone pAQ440. (Note *RBWallance et al Nucleic Acid
Res 9 879 (1981)) The nucleotide sequence of this (positive) clone pAQ440 was determined by the Maxam-Gilbert method, and the amino acid sequence and other protein properties were compared with those of the jellyfish photoprotein. Example 2 cDNA clone cloned in Example 1
The method for producing aquarin protein using pAQ440 is as follows. E. coli strain DH1 containing the cDNA clone pAQ440 plasmid in ampicillin, LB medium containing Bactotryptone (10 g), yeast extract (5 g), NaCl (5 g) in 1 part water at a concentration of 50 μg/ml;
PH7.2) and culture at 37℃ overnight. after that,
Harvest 10 ml of cultured bacteria by centrifugation at 5000 rpm for 10 minutes. Collect bacteria in 30mM Tris-
Dissolve in 1 ml of buffer containing HCl, PH7.6, and 10 mM EDTA, disrupt the bacterial cells by sonication (six times at 10 second intervals using a Branson Sonifer), and collect the supernatant by centrifugation at 10,000 rpm for 10 minutes. get. The aquarin activity of this supernatant is measured. The measurement composition was as follows: supernatant liquid 100μ buffer (30mM Tris-HCl, PH7.6, 10mM
M EDTA) 900 μ Coelenterazine (1 μg/ml) 5 μ 2-mercaptoethanol (manufactured by Wako) 5 μ, and after standing at 4°C for 2 hours, add 1 ml to this 100 μ
CaCl ₂ (30 mμ) is added, and the generated luminescence is detected photographically. As a control, pBR322 plasmid containing no aquarin gene was used to detect its luminescence. The results are shown in the table below.

[Table] It was clearly shown that the cDNA clone pAQ440 was used to produce an aquarin protein with luminescent ability.

[Brief explanation of drawings]

FIG. 1 shows the nucleotide sequence contained in the plasmid of the invention. FIG. 2 shows a restriction enzyme map of the aquarin gene of the cloned plasmid pAQ440. FIG. 3 shows a detailed restriction enzyme map of the cloned aquarin gene and the direction of nucleotide sequence determination by the Maxam-Gilbert method. FIG. 4 1 is a process chart showing a method for creating a cDNA library, and FIG. 4 2 is a process chart showing a method for screening an aquarin gene from a cDNA library. Figure 5 1 is the Okayama-Berg Vector
Figure 5 2 shows the preparation method for Oligo (dG) tailed
The method for preparing linker is shown.

Claims (1)

[Scope of Claims] 1. A plasmid having a substantially isolated complete gene specifying the biosynthesis of the jellyfish calcium photoprotein aquarin, and having the following deoxyribonucleotide sequence of the photoprotein aquarin. [Table] where the 5'-3' strand begins with an amide terminus and an amino acid with each triplet number indicated, and in each triplet, A is deoxyadenyl, T is thymidyl, and G is deoxy is guanyl, C is deoxycytosyl, Gly is glycine, Ala is alanine, Val is valine, Leu is leucine, Ile is isoleucine, Ser is serine, and Thr is Tyrosine, Trp is tryptophan, Cys is cysteine, Met is methionine, Asp is aspartic acid, Glu is glutamic acid, Lys is lysine, Arg is arginine, His is histidine. , Pro is proline, Gln is glutamine, and Asn is asparagine. 2. The DNA transport vector according to claim 1, which is transported to a microorganism and replicated by the microorganism. 3. The DNA transport vector according to claim 2, wherein the microorganism is a bacterium. 4 The bacteria is E. coli HB101 or DH1,
The plasmids are pBR322-SV40 (Okayama-Berg
The DNA transport vector according to claim 2, which is a vector).