HCV5’－NCR序列克隆和转录重组质粒构建

MOLECULAR CLONING OF HCV 5’NCR SEQUENCE AND ITS TRANSCRIPTABLE PLASMID RECOMBINATION

选取ＩＶ基因型ＨＣＶ及中国和台湾代表株ＨＣＶ的５’ＮＣＲ区保守序列合成引物，以ＲＴ－ＰＣＲ从输血后非甲非乙型肝炎病人血浆中扩增一段３００ｂｐ的ｃＤＮＡ片段作目的基因，钝端插入ｐＵＣ１９的ＳｍａＩ切点，构建了ｐＵＨＣＶ－ＮＣ重组质粒。对ｐＵＨＣＶ－ＮＣ分别或混合应用ＨＣＶ序列特异的内外引物和载体序列特异的通用引物进行ＰＣＲ扩增，所用产物分子量均同预期相符；酶切分析查出ＨＣＶ基因所带有和克隆位点融合所产生的两个外源ＮｃｏＩ切点也存在；证实ｐＵＨＣＶ－ＮＣ中克隆基因是ＨＣＶ的５’ＮＣＲ序列且插入方向为反向。应用ＢａｍＨＩ和ＥｃｏＲＩ双切出克隆ＨＣＶ基因，粘端插入ｐＳＰ７２的多克隆位点，均建了ｐＳＨＣＶ－ＮＣ转录重组体，对之进行了ＰＣＲ和酶切鉴定。

Form published data of five genotypes and two China strains of HCV,the conservative sequences in 5'NCR were applied to synthesize a pair of primers.By RT PCR,a cDNA fragment of 300 bp was amplified from plasma of a patient with post transfusion non A,non B hepatitis.By blunt ligation,the fragment was inserted reversely into the Sma Ⅰ site of the vector pUC19,and a recombinant plasmid pUHCV NC was generated.For identification of the insert,a series of testing PCR has been performed using two nested primer pairs specific to HCV,a primer pair specific to pUC19,and 4 mixed primer pairs from the above.In each reaction,an anticipated product was yielded that proves the insert to be 5'NCR sequence with reverse direction.Result of restrictive site analysis also supports it in which two exo pUC NcoI sites proved to be 257bp apart,one carried in target and another fused by ligation.The insert was then cut aside by Bam HI and EcoRI and cloned into the polyclonal sites of pSP72 to generate a recombinant pSHCV NC which was also determined by PCR and restrictive site analysis as descried.