狂犬病毒CVS-N2c毒株糖蛋白基因的克隆和重组腺病毒的构建  被引量:5

Cloning of glycoprotein cDNA of rabies virus CVS-N2C and the construction of recombinant adenovirus

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作  者:张云[1] 李文辉[1] 王树惠[1] 刘力[1] 杨帆[1] 

机构地区:[1]中国医学科学院中国协和医科大学基础医学研究所,北京100005

出  处:《基础医学与临床》2003年第3期332-336,共5页Basic and Clinical Medicine

基  金:国家 8 6 3计划项目 (86 3 10 1 0 5 0 4 0 3)

摘  要:克隆了狂犬病毒CVS N2c毒株糖蛋白 (GP)基因 ,并进行了GP全基因序列测定和对比分析。结果显示CVS N2cGP基因编码区长 15 75bp ,编码 5 2 4个氨基酸 ,与国内外发表的狂犬病毒疫苗株 3aG株、ERA株、和HEP Flury株GP基因的核酸序列同源性分别为 89 0 % ,89 3% ,94 5 % ,而推导的氨基酸序列同源性分别为 87 6 %、88 4 %、和91 2 %。在此基础上 ,用pAdEasy载体系统构建了表达GP基因的重组腺病毒。电镜下确定重组病毒颗粒的直径约70nm ,Westernblot显示重组病毒表达的GP蛋白可被兔抗狂犬病毒血清识别 ,表达的GP蛋白分子量约为 6 6kD ,与天然的狂犬病毒GP的分子量相符。该表达CVS N2c毒株GP基因的重组腺病毒是筛选有效狂犬病毒基因工程疫苗的基础。To clone the cDNA of glycoprotein gene(GP) of rabies virus CVS N2C strain and to construct a recombinant adenovirus bearing gp gene by pAdEasy system. The brain tissue of a rabies mouse was used to isolate viral RNA ,the cDNA of glycoprotein gene was obtained by RT PCR, followed by amplification of DNA fragment with high fidelity PCR system. The coding sequence of the glycoprotein gene was cloned into pUC18 plamid and then sequenced. The sequence was accepted by Genbank with an accession number of AF325714. Megalign analysis indicated that this glycoprotein gene was derived from rabies CVS_N2c, which was a variant of CVS24 strain with high neuropathogenicity. The gene had 1575 bp and coded for 524 amino acids. When compared to the glycoprotein gene of fixed strain of 3aG,ERA, HEP_Flury, CVS_N2cglycoprotein gene showed a similarity of 87.6%.88.4%, 91.2% and 87.6% 88.34%,91.2% similarity of deduced amino acid sequence was concluded respectively.The resultant recombinant adenovirus showed typical adenovirus morpholog with a diameter about 70nm.The expresed glycoproten by recombinant adebirus had a molecular mass 66kD which was similar to that of natural glycoprotei of rabie virus.

关 键 词:狂犬病毒 CVS—N2c毒株糖蛋白 基因克隆 基因序列测定 对比分析 重组腺病毒 狂犬病 

分 类 号:R346[医药卫生—基础医学]

 

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