汉滩病毒西安分离株84FLiM片段的克隆

Cloning of full-length complementary DNA of M gene of 84FLi, a Hantaan virus isolated from Xi'an

目的:克隆汉滩病毒84FLi株M片段的cDNA.方法:提取病毒总RNA,反转录为cDNA模板,用高保真的Taq酶扩增出G1和G2两个糖蛋白编码的基因片段:84M2.0(1～2058)和84M1.6(1964～3616).将这两个片段分别与T载体pMD18T连接,转化感受态大肠杆菌后获得两个分别为G1和G2编码的克隆pMD84M2.0和pMD84M1.6.再利用酶切连接的方法获得M片段全序列的cDNA克隆.结果:获得两个分别为G1和G2糖蛋白序列编码的克隆,进而酶切组装为含有M片段全序列的cDNA克隆.结论:获得了84FLi株M片段全序列的cDNA克隆.

AIM： To clone the full-length complementary DNA of M gene of 84FLi, a Hantaan virus. METHODS ： Total viral RNA was extracted and complementary DNA was synthesized by reverse transcription as template for PCR. Two PCR products - 84M2.0（ 1 - 2058） and 84M1.6（ 1964 - 3616）, containing coding region for G1 and G2,respectively,were inserted into pMD18T vector. Recombinant pMD84M2.0 and pMD84M1.6 were then transformed into competent E. coli. CDNA clone containing the full-length complementary DNA of M gene was obtained by enzyme digestion and ligation. PCR and restriction analysis were performed to identify the positive clone. RESULTS：Two cDNA clones containing the coding region for G1 and G2 were constructed. Then cDNA clone of the full-length M gene was obtained. CONCLUSION：The full-length complementary DNA of M gene of 84FLi was cloned successfully.