Expression of hepatitis C virus core protein deleted amino acid 60-80 in eukaryotic cell line

    HONG Sha£¬HUANG Changª²Xing£¬YANG Weiª²Song£¬CHEN Hongª²Mei£¬LI Guangª²Yu£¬WANG Pingª²Zhong£¬CHEN Weiª²Hong£¬ ZHANG Yan£¬LI Yu£¬BAI Xueª²Fan.

    Center of Diagnosis & Treatment for Infectious Diseases of PLA£¬Tangdu Hospital£¬Fourth Military Medical University£¬Xi¬ðan 710038£¬China

    ¡¾Abstract¡¿ AIM£º To construct an eukaryotic cell expression vector encoding a mutation hepatitis C virus(HCV) core protein deleted amino acids 60ª²80 and express it in p815 cell. METHODS: HCV core protein cDNA was amplified by RTª²PCR from blood serum of chronic hepatitis C patients. The gene fragment of mutation core(C510) was amplified by splicing by overlap extension(SOE) from the core protein cDNA and cloned into pCIª²neo, and named as pCIª²C510. By aid of lipofectamine2000,the expression vector was transfected into p815 cell. The expression protein was detected by immunofluorescence under confocal microscopy and westernª²blot. RESULTS: Phylogenetic analysis shows that the HCV core protein cDNA amplified from Shaanxi chronic hepatitis C patients were genotype 1 b.The pCIª²C510 was identified by sequencing. The signals of C510(p170) were diffusive in the cytoplasm under confocal microscopy.p170 was proved to be expressed in p815 cells by Western Blotting.CONCLUSION: The recombinant pCIª²C510 is correctly constructed and proved to be expressed effectively in p815 cells. ......