【作者】 满朝来；

【导师】 李一经；

【作者基本信息】 东北农业大学 ， 预防兽医学， 2003， 硕士

【摘要】 猪瘟是危害养猪业的主要传染病，为了得到大量的猪瘟病毒特异性诊断抗原，并试图在真核细胞中大量表达猪瘟病毒特有的免疫保护性抗原，即E2囊膜糖蛋白，为进一步制备猪瘟亚单位疫苗有效预防本病奠定基础。本试验首次克隆了我国地方分离株HL-LY E2囊膜糖蛋白基因，将该基因整合到毕赤酵母表达系统，使该基因获得了高效表达，并在此基础上探索了不同外界条件对表达量的影响，主要结果如下： 根据基因库已发表的猪瘟病毒E2基因序列及毕赤酵母表达载体pPIC9的多克隆位点序列，利用Oligo和Primer5.0等软件设计并合成一对引物，以提取猪瘟病毒HL-LY株总RNA为模板，应用RT-PCR技术，扩增得到E2基因。将该基因片段先克隆到pMD18-T载体上，并进行了酶切、PCR鉴定和测序分析，阳性重组质粒命名为T-E2。运用DNAMAN软件将测序结果与国内外已发表的猪瘟病毒Alfort株，Brescia株，SHIMEN株，C株，C-V-LZ株，GS-LT株和GS-LX株的E2基因全序列和E2基因主要抗原区序列(23bp～545bp)的核苷酸和氨基酸序列进行了同源性比较，E2基因全序列核苷酸的同源性分别为：99.20％，95.44％，96.51％，95.53％，89.12％，78.66％，78.23％；主要抗原区核苷酸的同源性分别为：98.90％，95.05％，96.70％，96.15％，95.42％，82.42％，83.15％。E2基因全序列氨基酸的同源性分别为：98.93％，95.98％，97.32％，95.71％，89.54％，83.16％，83.42％；主要抗原区氨基酸的同源性分别为：98.35％，95.05％，97.25％，95.60％，95.05％，87.36％，88.46％。结果表明E2基因主要抗原区和全序列均具有较高的保守性，与国内外代表毒株Alfort株，Brescia株，SHIMEN株，C株同源性较高，而与国内其他毒株C-V-LZ株，GS-LT株，GS-LX株同源性较低，说明我国部分地区猪瘟病毒存在E2基因突变株。 将重组质粒T-E2的E2基因酶切后，亚克隆到表达载体pPIC9上，经酶切和PCR鉴定，命名为pPIC9-E2。重组表达载体pPIC9-E2经Sal Ⅰ酶切线性化后，电转化整合到毕赤酵母GS115基因组上，经PCR鉴定和PCR产物测序分析，阳性转化子命名为GS115-pPIC9-E2。 将GS115-pPIC9-E2在30℃条件下振荡培养，以1％的甲醇诱导，猪瘟病毒E2融合蛋白获得了表达，表达量为0.34g／L。表达产物经SDS-PAGE和Western-blot和Dot-ELISA分析，确定其表达的融合蛋白分子量大小为50-54ku，且具有天然蛋白的抗原特异性。 通过对表达条件如pH值、通气量、诱导甲醇终浓度、培养基的选择等条件的探索，可确定猪瘟病毒HL-LY株E2基因表达的理想条件为：最适pH值在4.0～6.0之间，最适培养基为MM培养基，最佳诱导的甲醇终浓度为1％，加大通气量时表达量较好。更多还原

【Abstract】 Classical Swine Fever(CSF) is one of major contagious diseases in pig breeding industry. In order to gain massive diagnostic antigen CSFV, and large scale of CSFV specific major protective antigen protein(E2 envelope glycoprotein) in eucaryotic cells, which are prepared into subunit vaccine for effective prevention against CSF. E2 gene from CSFV HL-LY isolate was cloned and expressed in Pichia Pastoris expression system, moreover, conditions were explorerd for expressing the E2 envelope glycoprotein. The results are showed as follows.According to CSFV’s E2 gene sequence published in GenBank and the sequence of P.Pastoris expression vector pPIC9’s Multiple Cloning Site(MCS), a pair of primers were designed with Oligo and PrimerS.O softwares. E2 gene was amplified by RT-PCR from HL-LY isolate. The product of RT-PCR was cloned to pMD18-T vector, and identified by restriction endonuclease, PCR and sequencing, which was named as T-E2.The E2 gene’s sequence was analyzed by homology comparing with published E2 gene of CSFV Alfort strain, Brescia strain, SHIMEN strain, C strain, C-V-LZ strain, GS-LT strain and GS-LX strain.The homology of E2 gene complete sequence shares 99.20%, 95.44%, 96.51%, 95.53%, 89.12%, 78.66% and 78.23% in nucleotide respectively and shares 98.93%, 95.98%, 97.32%, 95.71%, 89.54%, 83.16% and 83.42% identities in amino acid sequence respectively.The major antigenic region of the sequence was also analyzed by homology comparing, which shares 98.90%, 95.05%, 96.70%, 96.15%, 95.42%, 82.42%, and 83.15% in nucleotide respectively and shares 98.35%, 95.05%, 97.25%, 95.60%, 95.05%, 87.36% and 88.46% identities in amino acid sequence respectively.The result shows that E2 gene is higher conservative, but there exist CSFV E2 gene mutation strains in China according to its lower homologies with C-V-LZ , GS-LT and GS-LT strains which were isolated from the different regions in China.E2 gene was cut with restriction endonuclease from the recombination plasmid T-E2, and subcloned into the expression vector pPIC9’s MCS, which was identified by enzyme digestion and PCR amplification. The recombination expression vector, pPIC9-E2, was linearized by Sal I and electroporated into P.Pastoris GS115. Recombinant P.Pastoris GS115, designated GS115-pPIC9-E2, was identified by PCR and the product of the PCR was analyzed by sequencing before its expression for CSFV E2 protein.Expression of CSFV E2 fusion protein was followed in Recombinant P.Pastoris GS115-pPIC9-E2 with the induction of 1% methanol at 30 C, shaking at 200rpm. A strong protein band of molecular mass estimation 50-54ku with SDS-PAGE was seen. This protein was detected on Western blots and Dot-ELISA probed with anti-CSFV serum, but P.Pastoris GS115 without recombinant E2 showed no protein in this region. It was estimated that theyield of the E2 fusion protein in culture supernatant of Recombinant P.Pastoris could be reached 0.34g/L.The optimal expression conditions were explored for the pH, aeration rate, final concentration of methanol and kinds of growth media. We conclude that the higher quantity of E2 protein can be gained at pH4.0-6.0, 1% methanol as the final concentration, MM growth media and high aeration rate.Candidate:Man Chaolai Speciality:Preventive Veterinary Science Supervisor:Prof. Li Yijing更多还原