| It is estimated that 3% population worldwide was infected with Hepatitis C virus (HCV), which is the major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. The development of prevention and therapy for HCV was hampered by the lacking of robust cell culture system and suitable small animal models. It is still unclear on the molecules which related to HCV recognition, attachment, and cell entry. It should be noted that the first step of viral infection and propagation is the binding of the virus on surface molecules of host cell, which was called receptor. Receptor is also the main factor for determining virus host specificity, tissue-tropism and pathogenicity. Several receptors and co-receptors, including CD81, SR-BI and Claudin-1, have been proved to induce HCV entry. Tree shrew (Tupaia belangera) is a small and quickly-bred animal, lives widely in the Southwest of China and Southeast of Asia. It's susceptibility by HCV have been proved by some preliminary results previously. The understanding of HCV receptor on hepatocytes of tree shrew could be used to deduce the pathogenicity of HCV, and provide new clues for finding new anti-virus compounds.In present study, the total RNA was extracted from liver tissue of tree shrew. CD81,SR-BI and Claudin-1 genes was amplified with Reverse Transcription, and then Polymerase Chain Reaction(RT-PCR). The produced bands were 711bp,1041bp and 636bp respectively. TA cloning technique was used to clone PCR product into pMD-18T vector. Then, sequencing and followed analysis was conducted. Sequence of amino acid of CD81,SR-BK Claudin-1 were deduced. Comparing with human beings, the similarity of nucleotide sequences were 91.4%, 87.5% and 90.9% respectively. Otherwise, the similarity of their amino acid sequence was 96.2%, 88.0% and 93.4%. Through the important amino acid analysis on CD81, there is total 9 mutated sites. Among them, 6 locate on Large Ectracellular Loop (LEL). There is the same amino acid between human being and tree shrew, which is necessary for HCV entry. Of Claudin-1, there is 14 mutated sites, and 3 located on LEL. Although there is more different amino acid in SR-BI, nucleotide and amino acid sequences of tree shrew is more similar with human being than other species.In order to quantitative detect the level of CD81 receptor in Tupaia hepatocytes, fluorescence real time PCR methods, which based on TaqMan technique, was developed. PCR primers and Taqman probe were designed and synthesized. Then, PCR system and procedure were optimized according to produced fluorescence level. Repetition and sensitivity of this method was evaluated. As a result, the linear relationship between the values of cycle threshold(Ct) and the copy number was significant (R~2 =0.9996). Its repetition is qualified (CV<5%). The higher sensitivity with low detection of 10~2 copies in 25μl reaction system could be found. This methods have provided a useful tool for CD81 quantitative detection. The decrease of CD81 RNA was found in HCV infected Tupaia hepatocytes with this methods.In summary, the phylogenetic analysis of receptor supports HCV susceptibility of Tupaia hepatocytes. The fluorescence quantitative PCR detection methods with high reproducibility, sensitivity and specificity, were established to detection CD81 RNA level. We initially explored the relationship between HCV infection and CD81 expression in PTH. The research on HCV receptors will be prompted by these findings.
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