【作者】 季新成；

【导师】 张彦明； 李晓成；

【作者基本信息】 西北农林科技大学 ， 预防兽医学， 2004， 硕士

【摘要】 本文利用反转录PCR（RT-PCR）和套式PCR（nPCR）技术，对流行于国内部分地区的9株猪瘟病毒（CSFV）E2基因进行克隆，并对其核苷酸及氨基酸序列的同源性进行比较及分析，绘制了系统关系发生树，以期了解近期猪瘟流行野毒主要保护性抗原E2基因的变异情况，为猪瘟的防制提供分子流行病学方面的资料。同时以1株（陕西周至株）为材料，在昆虫细胞中进行表达，为新型猪瘟基因工程亚单位疫苗的研制奠定了基础。（1）采用RT-PCR和nPCR扩增出9株国内近期猪瘟流行野毒株的E2基因，将其分别克隆于pMD18-T载体并进行核苷酸序列测定，根据C-株，Brescia株，Alfort株及Shime株确定起始氨基酸三联体的正确位置后进行氨基酸序列推导，并进行了同源性比较，绘制了系统关系发生树。结果表明,所测9株CSFV E2基因的长度均为1324 bp，编码的氨基酸序列均包括完整信号肽序列和跨膜序列，共由442个氨基酸残基组成。可将13株(包括4株参考毒株)CSFV 分为两个组群，GXGL ，GXNN，LNAS，SDJN 4株毒株与C 株，Shimen株， Alfort株和 Brescia株同为一个群组，其E2基因间的核苷酸序列同源性为94.0%～99.4%，所推导氨基酸序列同源性为93.7%～99.5%。HNLS ，SXZZ ，HNCA ，HNXY和 HNDX 5株同为另一群组，之间核苷酸序列同源性为81.9%～99.7%，所推导氨基酸序列同源性为88.2%～99.1%。13株E2基因间的核苷酸序列同源性为81.9%～99.7%，所推导氨基酸序列同源性为88.2%～99.5%，其中 9株流行野毒与4株参考毒株之间核苷酸序列同源性为81．9%～98.9%，所推导氨基酸序列同源性为88.2%～99.5%。说明近期流行毒株的变异呈现一定的多样性。通过对主要抗原区域氨基酸位点变异进行分析，发现各毒株间抗原决定簇未发生明显的变异。（2）对已知的E2基因进行分析后，用聚合酶链式反应（PCR）从陕西周至株克隆化E2全基因中扩增出除去3’端跨膜区的特异性片段（1 033 bp），并在其两端引入BamH I和Hind III酶切位点，将其亚克隆到杆状病毒转移载体pFastBacHTb，获得重组质粒 pFBHT-E2，转化进含穿梭载体Bacmid 的感受态细胞DH10Bac，得到含E2基因的重组载体质粒rBacmid-E2，以脂质体介导的方法将此重组载体质粒转染 sf9昆虫细胞，经SDS-PAGE和Western-blotting及ELASA等方法检测，结果表明，此E2基因在昆虫细胞中正确表达，表达的蛋白能与猪瘟阳性血清发生特异性反应。更多还原

【Abstract】 The paper compared the differation of E2 gene nucleotide of four reference strains and 9 prevalent strains classical swine fever virus (CSFV) ; the other is E2 gene of CSFV was expressed in insect cell．(1) The E2 gene of nine prevalent virulent strains of CSFV were amplified by reverse transcription polymerase chain reaction (RT-PCR) and the nested PCR(nPCR),Then them were cloned and sequenced, comparered with the published sequences of CSFV C-strain, Brescia，Alfort and Shime strain. The phylogenetic tree was constructed based on sequence comparion and analysis. The result indecated that there were at least two subgroups, GXNN, GXGL, LNAS , SDJN and C-strain, Brescia, Alfort, Shime strain all blonged to one subgrop. The nucleotide homology among them were 94.0% to 99.4%; and that of amino acid were 93.7% to 99.5%. HNLS, SXZZ, HNCA, HNXY and HNDX belonged to another subgroup. The nucleotide homology among them were 81.9% to 99.7％;and that of amino acid were 88.2% to 99.1%. The nucleotide homology of all the 13 strains were 81.9% to 99.7% also; and that of amino acid were 88.2% to 99.5%. It was showed epitopes of all strains were not variable obviously by analysising the variation of some main amino acid residues substitutions of E2 gene main antigenic domains．(2) Based on E2 gene sequence date, a fragment 1033bp in E2 gene (Guangxi Yuling Strain) cloned in plasmid pMD18-T was amplified by polymerase chain reaction(PCR).The E2 fragment was subcloned into baculovirus transfer vector and recombinant baculovirus vector pFBHT-E2 was extracted.Then it was transferred into E.coli DH10Bac and recombinant bacmid was constructed. The recombinant baculovirus was obtained after the tranferation of the recombinant bacmid into sf9 cell,Thetechniques of SDS-PAGE, Western-blotting and ELISA were used to detect and identify the products expressed in sf9 cell by recombinant baculovirus. The results indicated the E2 gene was properly expressed in sf9 cell, and the expressed protein could be recognized by the postive serum of CSFV.更多还原