CN109097366A - 突变的人2Ig-B7-H3蛋白编码基因、重组载体、其药物组合物及其应用 - Google Patents
突变的人2Ig-B7-H3蛋白编码基因、重组载体、其药物组合物及其应用 Download PDFInfo
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Abstract
本发明涉及基因工程领域，特别涉及一种突变的人2Ig‑B7‑H3蛋白编码基因，蛋白，重组载体，以及包括所述基因或蛋白的药物组合物。本发明的基因所编码的蛋白，其调节性表达、相互作用以及信号传递在肿瘤免疫应答过程中起着极其重要的作用，尤其为预防和治疗癌症提供了有利的新途径。
Description
技术领域
本发明涉及基因工程领域，特别涉及一种人2Ig-B7-H3蛋白编码基因、其编码的酶、重组载体、其药物组合物及应用。
背景技术
T细胞的活化需要两个不同的信号。第一信号来自TCR与抗原肽 -MHC复合物相互作用，第二信号来自APC上的B7家族分子与其在T 细胞上的配体CD28家族分子相结合产生的协同刺激信号，如B7-1B7-2 与CD28，CTLA-4结合，该途径被称为经典的B7途径。
人B7-H3基因首先是由Chapoval等在人类树突状细胞的cDNA文库中发现的，由于其结构类似B7家族的基因，因此命名为B7Homolog 3，简称B7-H3。它是一种Ⅰ型跨膜糖蛋白，属于免疫球蛋白超家族，在氨基酸序列上与B7家族的其他成员在细胞外有20-27％的同源性。
B7-H3有着广泛的表达：在转录水平B7-H3表达于大多数组织，在蛋白水平上仅表达于少数组织如人体肝、肺、膀胱、睾丸、前列腺、乳房、胎盘和淋巴样器官等；B7-H3在基因(mRNA)水平和蛋白水平上表达的差异可能与分子的转录后调控有关。
B7-H3除了在抗原特异性的体液免疫过程中能够调控淋巴细胞增殖，是一个免疫调控分子，近年来又发现它在许多肿瘤细胞中也有着重要的临床意义：即它在很多肿瘤细胞中都有高表达，可能是一个肿瘤抗性的调控因子。
人B7-H3基因定位在15号染色体上，蛋白在体内有两种不同形式的剪切体：2IgB7-H3和4IgB7-H3。2IgB7-H3胞外段由IgV-IgC两个免疫球蛋白结构域组成。申请人希望通过研究发现，2IgB7-H3的基因突变与肿瘤抗性的相关性。
发明内容
本发明要解决的技术问题是提供一种人2Ig-B7-H3蛋白编码基因、其编码的酶、重组载体、其药物组合物及应用，所述人2Ig-B7-H3蛋白编码基因对癌症的治疗提供了新的途径。
本发明公开了一种突变的人2Ig-B7-H3蛋白编码基因，所述基因是：
(1)SEQ ID NO:1所示的核苷酸序列。
本发明公开了一种人2Ig-B7-H3蛋白，所述人2Ig-B7-H3蛋白的编码基因是所述突变的人2Ig-B7-H3蛋白编码基因。
本发明公开了一种重组载体，所述重组载体包括载体及其携带的目的基因，所述目的基因是上述技术方案所述突变的人2Ig-B7-H3蛋白编码基因。
优选的，所述载体选自由克隆载体、真核表达载体、原核表达载体和穿梭载体。
本发明公开了一种药物组合物，所述药物组合物包括赋形剂以及选择上述技术方案所述人2Ig-B7-H3蛋白和所述的重组载体中的一种或几种。
优选的，所述药物组合物为注射液，所述注射液包括药学上可接受的赋形剂以及选自上述技术方案所述的重组载体中的一种或几种。
本发明公开了上述技术方案所述突变的人2Ig-B7-H3蛋白编码基因在制备预防和治疗癌症的药物中的应用。
与现有技术相比，本发明提供了一种突变的人2Ig-B7-H3蛋白编码基因，蛋白，重组载体，以及包括所述基因或蛋白的药物组合物。本发明的基因所编码的蛋白，其调节性表达、相互作用以及信号传递在肿瘤免疫应答过程中起着极其重要的作用，尤其为预防和治疗癌症提供了有利的新途径。
具体实施方式
下面将更详细地描述本公开的示例性实施例。本公开的示例性实施例可以以各种形式实现本公开而不应被这里阐述的实施例所限制。相反，提供这些实施例是为了能够更透彻地理解本公开，并且能够将本公开的范围完整的传达给本领域的技术人员。
本发明的实施例提供了一种突变的人2Ig-B7-H3蛋白编码基因，所述基因是：
(1)SEQ ID NO:1所示的核苷酸序列。
所述突变的人2Ig-B7-H3蛋白编码基因共包含2765个碱基，位于5’端第1488个碱基，发生C＞T杂合突变。
本发明公开了一种人2Ig-B7-H3蛋白，所述人2Ig-B7-H3蛋白的编码基因是所述突变的人2Ig-B7-H3蛋白编码基因。
本发明的实施例公开了一种重组载体，所述重组载体包括载体及其携带的目的基因，所述目的基因是上述技术方案所述突变的人2Ig-B7-H3蛋白编码基因。
其中，所述目的基因还可以包括调控序列，例如所述一种或几种目的基因表达的启动子、终止子和增强子。所述目的基因也可以包括标记基因(例如，编码β-半乳糖苷酶、绿色荧光蛋白或其它荧光蛋白的基因)或其产物调节其它基因表达的基因。所述目的基因除可以是DNA外，还可以是mRNA、tRNA 或rRNA，还可以包括通常与转录序列相关的相关转录调控序列，例如转录终止信号、聚腺苷酸化位点和下游增强子元件。
所述载体可以是本领域常用的各种能携带目的基因的载体以及技术发展改进的可用的各种能携带目的基因的载体。所述载体例如，质粒(裸DNA)、脂质体、分子耦联体、多聚物和病毒。
所述质粒(裸DNA)可以携带目的基因，该携带有目的基因的质粒可以直接注射或通过基因枪、电穿孔及电融合技术导入到组织细胞中。此外，超声波有助于提高质粒的转移效率。超声波配合微泡回声比差剂可提高细胞膜的通透性，从而显著提高裸DNA的转移和表达效率。这项胞膜渗透技术可在细胞膜表面瞬间制造小孔，DNA则趁机进入细胞内。
所述脂质体是由脂质双分子层组成的颗粒，可介导目的基因穿过细胞膜。所述脂质可以是来源于蛋黄和大豆的以卵磷脂(磷脂酰胆碱，PC)为主的天然磷脂；也可以是二棕榈酰磷脂酰胆碱(DPPC)、二棕榈酰磷脂酰乙醇胺(DPPE)、二硬脂酰磷脂酰胆碱(DSPC)等合成磷脂；还可以含有胆固醇。优选的脂质体为阳离子脂质体，其主要由带正电荷的脂类及中性辅助脂类以等摩尔混合而成。该带正电荷的脂质体与带负电荷的DNA之间可有效地形成复合物，并通过内吞作用移入细胞内。
所述多聚物，即利用阳离子多聚体，如多聚左旋赖氨酸上的正电荷与DNA 上的负电荷结合发生电性中和胍，而形成稳定的多聚物/DNA复合物。所得阳离子多聚体与DNA的复合物仍带正电荷，可与细胞表面带负电荷的受体结合，而被渗入至细胞内。
所述分子耦联体是将目的基因外源性DNA共价结合到细胞表面特异受体的配基或单克隆抗体或病毒胞膜蛋白上，利用特异的结合特性介导外源性基因导入至特定类型的细胞中。
病毒通常可以高效率地进入特定的细胞，表达自身蛋白，产生新的病毒粒子，因此被改造的病毒首先成为了基因治疗的载体。例如，反转录病毒载体、腺病毒载体、腺相关病毒载体及单纯疱疹病毒载体等。其中，所述腺相关病毒属于非致病性的微小病毒科家族成员，只有依赖于辅助病毒才可能增殖。腺相关病毒基因组很小，如2型腺相关病毒是由4681个核苷酸组成的单链DNA，包含2个基因，即rep基因(编码负责调节病毒复制、结构基因表达和整合到宿主基因组中的蛋白)及cap基因(编码衣壳结构蛋白)，基因组的1个末端存在1个145bp的末端重复区。腺相关病毒可感染分裂期及静止期细胞，能插入到宿主细胞染色体内，或以染色体外串联体DNA的形式长期稳定表达，可有效地转导脑、骨骼肌及肝脏等类型的细胞，具有抗原性、毒性小及不致病等特点。
优选的，所述载体选自由克隆载体、真核表达载体、原核表达载体和穿梭载体。
本发明的实施例公开了一种药物组合物，所述药物组合物包括赋形剂以及选择上述技术方案所述人2Ig-B7-H3蛋白和所述的重组载体中的一种或几种。
所述药物接受的赋形剂指无毒固态、半固态或液态填充剂、稀释剂、包囊材料或其他制剂辅料，例如，包括但不限于盐水、缓冲盐液、葡萄糖、水、甘油、乙醇及其混合物。所述药物组合物适于胃肠外、舌下、脑池内、阴道内、腹膜内、直肠内、颊内或表皮给药。
胃肠外给药包括静脉内、肌内、腹膜内、胸骨内、皮下、关节内注射和输注。适于胃肠外给药的药物组合物包括无菌水溶液或非水溶液、分散液、悬浮液或乳液，以及用于在临使用前在无菌可注射溶液或分散液中配制的粉末。适宜的水性或非水性载体、稀释剂、溶剂或赋形剂包括水、乙醇、甘油、丙二醇、聚乙二醇、羧甲基纤维素、植物油和可注射的有机酯如油酸乙酯。这些组合物还可以含有防腐剂、润湿剂、乳化剂、保护剂和分散剂佐剂，例如肌醇、山梨醇和蔗糖。优选加入渗透压调节剂如糖类、氯化钠、氯化钾。
表皮给药包括在皮肤、黏膜上以及在肺和眼表面给药。这样的药物组合物包括粉剂、软膏、滴剂、透皮贴剂、离子电渗疗法装置以及吸入剂等。直肠或阴道给药的组合物优选为栓剂，它可以通过将本发明的重组载体与适宜的非刺激性赋形剂如可可脂、聚乙二醇或栓剂蜡混合制备，所述赋形剂或载体在室温为固态，在体温下为液态，因此在直肠或阴道腔内熔化并释放出活性化合物。
优选地，所述药物组合物为注射液，所述注射液包括药学上可接受的赋形剂以及选自本发明所述的人葡萄糖激酶突变体编码基因和本发明所述的重组载体中的一种或几种。
优选的，所述药物组合物为注射液，所述注射液包括药学上可接受的赋形剂以及选自上述技术方案所述的重组载体中的一种或几种。
本发明的实施例公开了上述技术方案所述突变的人2Ig-B7-H3蛋白编码基因在制备预防和治疗癌症的药物中的应用。
以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域的普通技术人员来说，在不脱离本明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。
对所公开的实施例的上述说明，使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的，本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下，在其它实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
Claims (7)
1.一种突变的人2Ig-B7-H3蛋白编码基因，其特征在于，所述基因是：
(1)SEQ ID NO：1所示的核苷酸序列。
2.一种人2Ig-B7-H3蛋白，其特征在于，所述人2Ig-B7-H3蛋白的编码基因是权利要求1所述突变的人2Ig-B7-H3蛋白编码基因。
3.一种重组载体，所述重组载体包括载体及其携带的目的基因，其特征在于，所述目的基因是权利要求1所述突变的人2Ig-B7-H3蛋白编码基因。
4.根据权利要求3所述的重组载体，其特征在于，所述载体选自由克隆载体、真核表达载体、原核表达载体和穿梭载体。
5.一种药物组合物，其特征在于，所述药物组合物包括赋形剂以及选择权利要求2所述人2Ig-B7-H3蛋白和权利要求3～4任意一项所述的重组载体中的一种或几种。
6.根据权利要求5所述的药物组合物，其特征在于，所述药物组合物为注射液，所述注射液包括药学上可接受的赋形剂以及选自权利要求3或4所述的重组载体中的一种或几种。
7.权利要求1所述突变的人2Ig-B7-H3蛋白编码基因在制备预防和治疗癌症的药物中的应用。
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