人乳头瘤病毒16型晚期蛋白L1预防性疫苗的研究

发布时间：2019-03-27 09:15

【摘要】： 人乳头瘤病毒(HPV)感染属于严重的性传播疾病，给人类健康带来很大的影响，临床学、分子生物学和流行病学调查已经证明人乳头瘤病毒是宫颈癌的主要病因，并且随着科学技术的快速发展，分子生物学和基因工程技术的应用不断进步，预防和治疗宫颈癌的疫苗倍受许多科学家的重视。因此，此项疫苗的研究具有非常重要的社会效益和经济效益。 目前大多数关于HPV疫苗的研究都是以其晚期蛋白L1或L1+L2所形成的病毒样颗粒(VLPs)为基础进行的，在本实验中，我们应用毕赤酵母表达系统开展了基因工程HPV16L1预防性疫苗的研究工作，取得了一些进展，为该疫苗的进一步发展奠定了基础。实验期间主要得到了以下几个方面的结果： 1)搜索Genebank中有关HPV16L1的基因序列，共搜索到30条较完整的序列，用软件AlignX进行序列比对，确定同源性最大的氨基酸序列，再根据毕赤酵母密码子进行优化，，送上海生工合成，但三个月后送来两个片段，两片段间有一段重复序列(其中都含有BglⅡ的酶切位点)，将两个片段分别酶切后通过BglⅡ进行连接，将连接产物作为模板，进行PCR反应，将其产物进行鉴定，合成了全长为1536bp的HPV16L1基因。 2)构建了酵母重组质粒pAO819-16L1，经酶切鉴定正确后通过电击转化到酵母菌株GS115中，经G418抗性筛选获高拷贝工程菌并用甲醇(1％)诱导进行表达，每隔24h补加甲醇1％，在诱导后48h、72h和96h收菌，用Western Blot鉴定发现在55KD处出现特异性的条带，而阴性对照中未出现目的条带，发现诱导后72h表达量最高。 3)构建原核重组质粒pET43.1a-16L1，经酶切鉴定正确后转化至BL21 codonplus(DE3)和BL21 codonPlus(DE3)-RILP菌株中进行表达，目的蛋白以包涵体的形式存在，通过8M尿素变性处理后过离子交换树脂，分阶段进行洗脱，0.1MNaCl可洗脱杂蛋白，0.5MNaCl可洗脱目的蛋白，纯度可达80％以上，以便对酵母表达的目的蛋白进行阳性对照。 4)构建了真核重组质粒pDRVI1.0-16L1，经酶切和测序鉴定正确后，用试剂盒大提质粒免疫小鼠，共免疫四针，每次免疫后通过电穿孔加强免疫效果，制备了抗HPV16L1的抗血清，通过WB鉴定后发现该抗体能产生特异性的条带，滴度较高，但特异性不强。
[Abstract]:Human papillomavirus (HPV) infection is a serious sexually transmitted disease and has a great impact on human health. Clinical, molecular biology and epidemiological investigations have shown that human papillomavirus is the main cause of cervical cancer. With the rapid development of science and technology and the application of molecular biology and genetic engineering technology, the vaccine to prevent and treat cervical cancer has been paid more and more attention by many scientists. Therefore, the research of this vaccine has very important social and economic benefits. At present, most of the studies on HPV vaccine are based on the virus-like particles of L1 or L1 L2. In this experiment, we found that the virus-like particles formed by the late protein L1 or L1 L2 are the basis of this study. We applied Pichia pastoris expression system to the study of genetic engineering HPV16L1 prophylactic vaccine, and made some progress, which laid a foundation for the further development of the vaccine. During the experiment, the following results were obtained: 1) searching for the gene sequences of HPV16L1 in Genebank, searching 30 more complete sequences, and comparing the sequences with software AlignX. The amino acid sequence with the highest homology was determined and optimized according to Pichia pastoris dense code. It was sent to Shanghai Shenggong for synthesis, but three months later, two fragments were sent, and there was a repeat sequence between the two fragments (which both contained the cleavage site of Bgl 鈪

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