【作者】 韩雪清；

【导师】 张涌； 谢庆阁；

【作者基本信息】 西北农林科技大学 ， 临床兽医学， 2002， 博士

【摘要】 针对频繁出现的弱毒疫苗免疫失败现象和免疫猪群持续带毒等猪瘟(CSF)防制中的重大问题，本项研究从病毒的分子水平入手，试图从病毒分子变异水平以及当前流行毒的分子差异程度澄清原因，并针对CSF的持续感染研制出高效安全的基因工程标记疫苗和诊断试剂，为彻底消灭CSF提供科学依据和技术支撑。主要研究内容包括： 1．猪瘟病毒主要免疫原E2基因的序列分析：利用反转录PCR(RT-PCR)及套式PCR(nPCR)方法扩增了中国猪瘟兔化弱毒株(C株)兔脾组织毒保护性抗原E2(gp55)基因，成功地将其克隆并测定核苷酸序列，与国内外已发表的猪瘟病毒(CSFV)E2基因序列比较的结果是：C-株兔脾毒与C-株细胞(SK6)毒、C-株疫苗(犊牛睾丸细胞，CSFV-C)毒CSFV-SM株(石门)毒、Bresia株(荷兰)毒、Alfort株(德国)毒的E2核苷酸序列同源性分别为98.87％、98.34％、94.58％、91.00％、80.78％；氨基酸同源性分别为98.95％、97.37％、94.22％、91.60％、89.23％。对C-株兔脾毒与C-株细胞毒、经典强毒E2上的A、B、C三个中和性抗原区的氨基酸组成进行了比较，其结果为：C-株兔脾毒与C-株疫苗细胞毒的差异很小甚至没有差异。 2．猪瘟病毒分子流行病学分析：利用RT-PCR及nPCR扩增并测定了我国近期(1997～2001年)12个省的51株流行野毒株的E2基因核酸序列。通过序列比较和系统发生关系分析发现：C-株兔组织毒、C—株细胞毒和中国50～60年代流行的石门强毒株同属于组群1(Group 1)；近期猪瘟流行毒株均属于组群2(Group 2)的两个不同的亚组群(Subgroup2.1和2.2)，表明我国目前流行毒株至少有2个亚组群。流行毒株与疫苗毒株有较大的差异(E2全基因核苷酸序列同源性82.2％～84.3％)，表明猪瘟流行毒株已远离疫苗毒株的方向演变，明确了我国CSF流行趋势及我国CSFV在世界SCFV大系谱中的位置，并为基因工程疫苗的研究提供了基因储备。 3．猪瘟病毒E2基因的真核表达：分别将CSFV两个代表株的E2基因克隆入毕赤酵母(P.Pastoris)分泌型表达载体pPIC9K中，酶切线型化后电穿孔导入P.Pastotis进行整合，经G418筛选得到25个高拷贝转化子，经DNA斑点试验和DNA测序证明外源基因E2稳定地整合到P.Pastoris染色体中。经甲醇诱导表达后，SDS-PAGE和Western blot结果证明了P.Pastoris培养上清液中含有正确糖基化的E2蛋白，表达量约250mg/L。重组P.Pastoris经连续培养8代仍可分泌特异性蛋白。免疫活性研究证明P.Pastoris表达的E2蛋白能刺激动物产生1：128～1：256猪瘟病毒的抗体。有望用于基因工程亚单位疫苗的研制。 4．猪瘟病毒E2基因的密码子改造优化：低利用率密码子的数量和分布是影响外源基因有效表达的参数之一，优化密码子序列能够提高外源基因的表达。利用PCR基础上的定点突变方法对猪瘟病毒E2基因的24个密码子进行优化。优化后的E2基因转化P.Pastoris菌，甲醇诱导表达。诱导培养上清的SDS-PAGE和Western blot分析显示，E2蛋白在P.Pastoris中得到了高效的表达，表达量约1.08g／L。这表明对E2基因上低利用密码子的改造是成功的， 猪瘟病毒EZ基因遗传变异及体外表达研究为进一步大规模生产打下了基础。5．毕赤酵母高效表达的培养条件探索：用PPastoris系统表达外源基因，对于不同的外源蛋白，表达量千差万别。除外源基因序列本身的内在特性起着很重要的作用外，表达条件对表达量高低的影响也极其显著。分别在不同时间、不同诱导型、不同pH、不同诱导剂量方面进行了较系统的对比：经不同时间表达，表明72小时蛋白可达峰值；采用抑制／诱导方式会提高表达量；表达EZ蛋白pH最佳值在7．5—8刀；最佳甲醇诱导量是2％～3％；得到了一套较完善的在PPastoris中表达CSFV EZ基因的条件，以供在规模化发酵罐中表达EZ基因蛋白生产亚单位疫茵。6．猪瘟病毒EZ基因的原核表达：PCR扩增出当前猪瘟流行野毒株，中国猪瘟兔化弱毒（C－株）兔脾组织毒EZ基因的主要抗原区，将其克隆到原核大肠杆菌表达载体PPROEX－HTb中诱导表达，经 SDS－PAGE检测表明，重组质粒能表达EZ基因主要区蛋白，Western blot检测表明，诱导表达蛋白与猪瘟阳性血清发生特异性反应，表达量为35％和38％，可用于基因工程诊断抗原。7．猪瘟病毒EZ基因的乳腺特异表达载体构建：将在乳腺细胞中特异分泌的信号肽序列连接到EZ基因，PCR得到了目的片段，再将pGFPCI载体上的Kana基因切下与在乳腺细胞中特异表达的P22载体连接，使其带有筛选标记，然后将带有信号肽的EZ插入到P22中，试图构建山羊乳腺上皮细胞的特异性表达载体。探索了构建的含目的基因的乳腺特异性表达载体调控成分的有效性，也为在体外培养的乳腺细胞中定位重组目的基因一体细胞克隆一动物乳腺生物反应器工作的开展作准备工作。更多还原

【Abstract】 The E2 gene of CSFV C strain derived from rabbit spleen tissue was amplified by reverse transcription (RT) and nested Polymerase Chain Reaction (nPCR), then was cloned and sequenced. Compared with the published sequences of CSFV other virulent strains, the nucleotide homology of C strain from rabbit spleen tissue with strain C adapted to SK6 cell, Shimen, Brescia, Alfort strain were 98.87%, 94.58%, 91.00% and 80.78% respectively, and the amino acid homology were 98.95%, 94.22%. 91.60% and 89.23% respectively. There were little difference in the amino acid composition of antigen domain of A, B and C between C strain from rabbit spleen tissue and virulent strains above. This indicated that the prevalence of CSF and immunal failure may be not caused by the antigen variation of C stain vaccine virus.The E2 genes of 51 CSFV prevalent virulent strains isolated from 12 provinces in China were amplified and sequencd. The phylogenetic tree was constructed based on sequence comparison and analysis. The result showed that C strain derived from rabbit spleen tissue, C strain adapted to SK6 cell, Shimen virulent strain all belonged to group 1.1, and the prevalent virulent strains belonged to subgroup 2.1 or subgroup 2.2. This indicated there were at least two subgroups, which were prevailing in China, and the prevalent virulent strains were diverse far from C. strain vaccine virus.The E2 genes above of the prevalent Strain (Guangxi Yulin Strain) were cloned respectively into secreted expression vector pPIC9K of eukaryotic expression system P.Pastoris and transformed into P.Pastoris by electroporation after linearization, 25 high-copied transformants were obtained by G418 screening. It was proved that the E2 genes were integrated stably into chromosome of P.Pastoris by Dot blot and DNA sequencing. The results of SDS-PAGE and Western blot showed that the culture supernatant of transforments contained the correct glycosylated E2 protein and the recombinants could still secretedly express the specific proteins after culturing for 8generations. Studies on immune activity showed that the E2 protein expressed in P.Pastoris could induce animals to produce CSFV specific antibodies, the titre amounts to 1:128-1:256. This suggests proved that the E2 protein could protect animals against virulent virus of CSFV. Its the first time that the CSFV E2 protein was expressed successfully in eukaryotic expression system P.Pastoris.In order to improve the expression level of E2 gene in P.Pastoris, 24 low -usage codons for P.Pastoris were substitued by directed mutagnesis based on PCR. The optimized E2 gene was cloned into P.Pastoris expression vector pPIC9K and transformed into P.Pastoris SMD1168 host cell. The transformants were induced with methanol to express the interested gene .The results of SDS -PAGE and Western blot showed that E2 protein was expressed at high level, up to 1.08g/L.The expression conditions of E2 gene in P.Pastoris were optimized, the results indicated that the peak obtained after 72 hours;pattern of inhibition/ induction could improve expression level; The best pH value were between 7.5 and 8.0 and the optimized methanol-induced concentration was 2%-3%The E2 genes of the prevalent Strain (Guangxi Yulin Strain) and C strain derived from rabbit spleen tissue were amplified and cloned into E.coli the expression vector pPROEX-HTb respectively, the recombinant plasmids pPROEX-GXYL and pPROEX-C were obtained and then were transformed into the DH5a E.coli competent bacteria respectively, the recombinant bacteria could express the major antigen region of E2 gene, the expression yields amount to 35% and 38% repectively.The signal peptide sequence which was used for secretedly expressing divergent gene in mammary gland cells was ligated to the E2 gene, the E2 gene with signal sequence was obtained by PCR .The gene resisting kanamycin was cut down from pGFP-Cl vector and inserted into P22 vector, a P22 vector with gene resisting kanamycin was constructed, it was tried to construct an expression vector for trans更多还原