犬冠状病毒大熊猫株纤突蛋白全基因的克隆与序列分析

在国内首次对犬冠状病毒大熊猫野毒株(CCVDXMV)纤突蛋白基因进行了克隆和序列测定。该基因全长4362bp,编码1453个氨基酸,N端前18个氨基酸为推测的信号肽序列,后1435个氨基酸构成成熟蛋白。与GenBank中已发表的11个CCV毒株s基因相比,s基因核苷酸序列同源性在40．2％-99．5％之间;推导的氨基酸序列同源性在15．9％-99．O％之间。DXMV株s基因变异区主要集中在该基因前1／2处,其中350．370、439．478、1718．1818三个区域碱基变异较大,而1060．1700区却十分保守。基于s全基因及其蛋白的聚类分析表明,DXMV株与K378、NVSL和USpatent株亲源关系最近。推导的DXMV株s蛋白氨基酸序列潜在的N-联糖基化位点与CCV强毒V54相同,为34个,比Insavc．1弱毒多一个;其中第566．568位糖基化位点为多数强毒拥有而弱毒没有的。另外,DXMV株S蛋白疏水性及抗原表位与其它毒株有一定的差异,这些差异对DXMV株致病性和免疫原性等影响尚待进一步的研究。     

应用细菌内同源重组机制快速克隆腺病毒基因组的研究

A novel procedure was used for cloning large adenovirus genome fragment by the homologous recombination in E.coli strain BJ5183. The 11.2Kb downstream fragment of the CAV-2 strain YCA18 genome was cloned by homologous recombination, the 1029bp left end and the 970bp fight end of this fragment were separately amplified by PCR. They were then cloned into plasmid pPoly2 with direction from left fragment to fight fragment, obtaining a “rescue” plasmid pT615. The pT615 was liberalized by Hind Ⅲ and PstⅠ digestion and was cotransformed with the purified CAV-2 genome which was cut by BstBI into competent E.coli strain BJ5183. Recombinant plasmids harboring the 11.2Kb downstream fragment of CAV-2 genome were obtained after bacterial intermolecular homologous recombination. The recombinant efficiency of all E.coli strains tested was 78.3%. One of the recombinant plasmids, pT618, was further identified by enzyme digestion analysis and PCR amplification. The results showed the plasmids contained the 11.2kb fragment downstream the genome of CAV-2.