【摘要】： 人乳頭瘤病毒(HPV)感染屬于嚴重的性傳播疾病，給人類健康帶來很大的影響，臨床學、分子生物學和流行病學調查已經(jīng)證明人乳頭瘤病毒是宮頸癌的主要病因，并且隨著科學技術的快速發(fā)展，分子生物學和基因工程技術的應用不斷進步，預防和治療宮頸癌的疫苗倍受許多科學家的重視。因此，此項疫苗的研究具有非常重要的社會效益和經(jīng)濟效益。 目前大多數(shù)關于HPV疫苗的研究都是以其晚期蛋白L1或L1+L2所形成的病毒樣顆粒(VLPs)為基礎進行的，在本實驗中，我們應用畢赤酵母表達系統(tǒng)開展了基因工程HPV16L1預防性疫苗的研究工作，取得了一些進展，為該疫苗的進一步發(fā)展奠定了基礎。實驗期間主要得到了以下幾個方面的結果： 1)搜索Genebank中有關HPV16L1的基因序列，共搜索到30條較完整的序列，用軟件AlignX進行序列比對，確定同源性最大的氨基酸序列，再根據(jù)畢赤酵母密碼子進行優(yōu)化，，送上海生工合成，但三個月后送來兩個片段，兩片段間有一段重復序列(其中都含有BglⅡ的酶切位點)，將兩個片段分別酶切后通過BglⅡ進行連接，將連接產物作為模板，進行PCR反應，將其產物進行鑒定，合成了全長為1536bp的HPV16L1基因。 2)構建了酵母重組質粒pAO819-16L1，經(jīng)酶切鑒定正確后通過電擊轉化到酵母菌株GS115中，經(jīng)G418抗性篩選獲高拷貝工程菌并用甲醇(1％)誘導進行表達，每隔24h補加甲醇1％，在誘導后48h、72h和96h收菌，用Western Blot鑒定發(fā)現(xiàn)在55KD處出現(xiàn)特異性的條帶，而陰性對照中未出現(xiàn)目的條帶，發(fā)現(xiàn)誘導后72h表達量最高。 3)構建原核重組質粒pET43.1a-16L1，經(jīng)酶切鑒定正確后轉化至BL21 codonplus(DE3)和BL21 codonPlus(DE3)-RILP菌株中進行表達，目的蛋白以包涵體的形式存在，通過8M尿素變性處理后過離子交換樹脂，分階段進行洗脫，0.1MNaCl可洗脫雜蛋白，0.5MNaCl可洗脫目的蛋白，純度可達80％以上，以便對酵母表達的目的蛋白進行陽性對照。 4)構建了真核重組質粒pDRVI1.0-16L1，經(jīng)酶切和測序鑒定正確后，用試劑盒大提質粒免疫小鼠，共免疫四針，每次免疫后通過電穿孔加強免疫效果，制備了抗HPV16L1的抗血清，通過WB鑒定后發(fā)現(xiàn)該抗體能產生特異性的條帶，滴度較高，但特異性不強。
[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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