【摘要】： 人乳頭瘤病毒(HPV)感染屬于嚴(yán)重的性傳播疾病，給人類健康帶來(lái)很大的影響，臨床學(xué)、分子生物學(xué)和流行病學(xué)調(diào)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(xué)技術(shù)的快速發(fā)展，分子生物學(xué)和基因工程技術(shù)的應(yīng)用不斷進(jìn)步，預(yù)防和治療宮頸癌的疫苗倍受許多科學(xué)家的重視。因此，此項(xiàng)疫苗的研究具有非常重要的社會(huì)效益和經(jīng)濟(jì)效益。 目前大多數(shù)關(guān)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(chǔ)進(jìn)行的，在本實(shí)驗(yàn)中，我們應(yīng)用畢赤酵母表達(dá)系統(tǒng)開(kāi)展了基因工程HPV16L1預(yù)防性疫苗的研究工作，取得了一些進(jìn)展，為該疫苗的進(jìn)一步發(fā)展奠定了基礎(chǔ)。實(shí)驗(yàn)期間主要得到了以下幾個(gè)方面的結(jié)果： 1)搜索Genebank中有關(guān)HPV16L1的基因序列，共搜索到30條較完整的序列，用軟件AlignX進(jìn)行序列比對(duì)，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(jìn)行優(yōu)化，，送上海生工合成，但三個(gè)月后送來(lái)兩個(gè)片段，兩片段間有一段重復(fù)序列(其中都含有BglⅡ的酶切位點(diǎn))，將兩個(gè)片段分別酶切后通過(guò)BglⅡ進(jìn)行連接，將連接產(chǎn)物作為模板，進(jìn)行PCR反應(yīng)，將其產(chǎn)物進(jìn)行鑒定，合成了全長(zhǎng)為1536bp的HPV16L1基因。 2)構(gòu)建了酵母重組質(zhì)粒pAO819-16L1，經(jīng)酶切鑒定正確后通過(guò)電擊轉(zhuǎn)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(dǎo)進(jìn)行表達(dá)，每隔24h補(bǔ)加甲醇1％，在誘導(dǎo)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(duì)照中未出現(xiàn)目的條帶，發(fā)現(xiàn)誘導(dǎo)后72h表達(dá)量最高。 3)構(gòu)建原核重組質(zhì)粒pET43.1a-16L1，經(jīng)酶切鑒定正確后轉(zhuǎn)化至BL21 codonplus(DE3)和BL21 codonPlus(DE3)-RILP菌株中進(jìn)行表達(dá)，目的蛋白以包涵體的形式存在，通過(guò)8M尿素變性處理后過(guò)離子交換樹(shù)脂，分階段進(jìn)行洗脫，0.1MNaCl可洗脫雜蛋白，0.5MNaCl可洗脫目的蛋白，純度可達(dá)80％以上，以便對(duì)酵母表達(dá)的目的蛋白進(jìn)行陽(yáng)性對(duì)照。 4)構(gòu)建了真核重組質(zhì)粒pDRVI1.0-16L1，經(jīng)酶切和測(cè)序鑒定正確后，用試劑盒大提質(zhì)粒免疫小鼠，共免疫四針，每次免疫后通過(guò)電穿孔加強(qiáng)免疫效果，制備了抗HPV16L1的抗血清，通過(guò)WB鑒定后發(fā)現(xiàn)該抗體能產(chǎn)生特異性的條帶，滴度較高，但特異性不強(qiáng)。
[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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