本文選題：人乳頭瘤病毒11型 + L1蛋白　； 參考：《廈門大學(xué)》2007年碩士論文

【摘要】： 流行病學(xué)和相關(guān)研究證實(shí)人乳頭瘤病毒(HPV)感染與尖銳濕疣、宮頸上皮瘤樣增生和宮頸癌的發(fā)生關(guān)系密切。其中,HPV6、11型是尖銳濕疣的主要致病因子,大約90%的生殖器疣與HPV6和HPV11有關(guān)。預(yù)防HPV11感染的疫苗研制具有良好的社會(huì)效益及經(jīng)濟(jì)效益。 研究結(jié)果表明,HPV L1能獨(dú)立組裝成為HPV類病毒顆粒(VLP),從而良好地再現(xiàn)HPV的天然中和表位,并具有較好的免疫原性,是目前預(yù)防性疫苗研究的主要目標(biāo)蛋白。本研究利用原核表達(dá)系統(tǒng)非融合表達(dá)HPV11 L1蛋白及其突變體,研究N端序列以及關(guān)鍵氨基酸對HPV11 L1的表達(dá)和VLP組裝效率的影響,從而獲得可用于預(yù)防HPV11病毒感染的疫苗。 首先,HPV11-L1蛋白N端缺失突變結(jié)果表明,N端缺失不超過16aa的突變不影響VLP的形成,但對目的蛋白的表達(dá)量有不同程度的影響,其中缺失4aa的突變體HPV11N4C-L1在大腸桿菌中的表達(dá)量較大,可用于預(yù)防性疫苗的研究。 其次,分析Genebank中兩株HPV11序列發(fā)現(xiàn)在aa364、aa453、aa501出現(xiàn)差異(分別為Glu~(364)和Lys~(364)、Ser~(453)和Tyr~(453)、Lys~(501)和Arg~(501)),實(shí)驗(yàn)顯示兩種蛋白的理化性質(zhì)和VLP的組裝有較大的差異。定點(diǎn)突變實(shí)驗(yàn)表明,aa453與aa501的差異并不影響蛋白的性質(zhì),但aa364對VLP的組裝效率至關(guān)重要。進(jìn)一步將aa364突變成為Gln、Asp、Arg、Phe、Gly和Leu的實(shí)驗(yàn)表明,氨基酸側(cè)鏈對蛋白的表達(dá)和組裝有顯著的影響。HPV11 L1蛋白的結(jié)構(gòu)模建結(jié)果表明,aa364位于維系L1結(jié)構(gòu)的重要β-折疊上,計(jì)算機(jī)突變分析提示aa364側(cè)鏈參與的氫鍵形成以及與周圍氨基酸的碰撞可能是影響VLP組裝的原因。多HPV序列比對分析顯示,相當(dāng)于HPV11-L1 aa364的氨基酸在其它型別序列上高度保守,均為本研究所確認(rèn)的Glu。 最后,利用上述本研究的突變研究,選定HPV11N4C-L1(含Glu~(364))為目的基因進(jìn)行HPV11預(yù)防性疫苗的研制。本研究成功地建立了一套發(fā)酵、純化和復(fù)性工藝,并獲得達(dá)到新藥臨床試驗(yàn)要求的HPV11型疫苗。動(dòng)物實(shí)驗(yàn)結(jié)果顯示,該疫苗可誘導(dǎo)小鼠產(chǎn)生較高滴度的中和抗體,中和抗體與HPV6型存在明顯的交叉反應(yīng)。 總之,本研究為HPV11的L1蛋白性質(zhì)研究、VLP組裝機(jī)理提供了結(jié)構(gòu)信息,為HPV11疫苗的分子設(shè)計(jì)提供了參考,成功地研制出針對HPV11的預(yù)防性疫苗,并為HPV6和HPV11雜合疫苗的研制提供了依據(jù)。
[Abstract]:Epidemiology and related studies have confirmed that HPV infection is closely related to condyloma acuminatum, cervical epitheliomatous hyperplasia and cervical cancer. HPV6 type 11 is the main pathogenic factor of condyloma acuminatum. About 90% of genital warts are related to HPV6 and HPV11. The development of vaccine to prevent HPV11 infection has good social and economic benefits. The results show that HPVL1 can be assembled independently to form HPV virus-like particles, thus the natural neutralizing epitopes of HPV can be reproduced well and have good immunogenicity, which is the main target protein of prophylactic vaccine research at present. In this study, the prokaryotic expression system was used to express HPV11 L1 protein and its mutants. The effects of N-terminal sequence and key amino acids on HPV11 L1 expression and VLP assembly efficiency were studied in order to obtain a vaccine for the prevention of HPV11 virus infection. First of all, the N-terminal deletion mutation of HPV11-L1 protein showed that the N-terminal deletion of HPV11-L1 protein did not affect the formation of 16aa, but had different effects on the expression of target protein. The mutant HPV11N4C-L1 with deletion of HPV11-L1 had a higher expression level in Escherichia coli. It can be used in the study of prophylactic vaccine. Secondly, the two HPV11 sequences in Genebank were found to be different in Aa364A453A501 (Glufus 364) and Lysnus 364Sequin453 and Tyrrhium 453 (Lysxanthine 501) and Arg501. the results showed that the physicochemical properties of the two proteins and the assembly of VLP were quite different from those of the two proteins. The results showed that there were significant differences between the two HPV11 sequences and that between the two HPV11 strains. The results showed that there were significant differences in the physical and chemical properties of the two proteins and the assembly of VLP. Site-directed mutagenesis test showed that the difference between A453 and aa501 did not affect the properties of the protein, but aa364 was very important to the assembly efficiency of VLP. Furthermore, the mutagenesis of aa364 into Glnsil-Asp-Argopheus gly and Leu showed that the side chain of amino acid had a significant effect on protein expression and assembly. The result of structural modeling of HPV11 L1 protein showed that Aa364 was located on the important 尾 -fold that holds the L1 structure. Computer mutation analysis suggested that the formation of hydrogen bonds in the side chain of aa364 and the collision with the surrounding amino acids might be the factors affecting the assembly of aa364. Multiple HPV-sequence alignment analysis showed that the amino acids corresponding to HPV11-L1 aa364 were highly conserved in other types, all of which were confirmed by our study. Finally, HPV11N4C-L1 was selected as the target gene for the preparation of HPV11 prophylactic vaccine. In this study, a set of fermentation, purification and renaturation processes were successfully established, and HPV11 vaccine was obtained to meet the requirements of new drug clinical trials. The results of animal experiments showed that the vaccine could induce higher titer of neutralizing antibody in mice, and there was obvious cross reaction between neutralizing antibody and HPV6 type. All in all, this study provides structural information for the study of L1 protein properties of HPV11 and provides a reference for the molecular design of HPV11 vaccine. A prophylactic vaccine against HPV11 was successfully developed. It also provides the basis for the development of hybrid vaccine of HPV6 and HPV11.
【學(xué)位授予單位】：廈門大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R392

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