应用In—Fusion技术构建HBV基因组全长重组质粒及序列分析

Use of In-Fusion technology in cloning of the full-length genome of hepatitis B virus and phylogeneticanalysis

目的研究In—Fusion技术在HBV全进组克隆中的应用，并分析所获得的的序列信息。方法在浙江杭州地区收集23份慢性乙型肝炎患者血清，提取病毒核酸，分别设计PCR引物扩增病毒基因组和pMDl8T载体，采用In-Fusion方法构建基因组测序质粒，并对序列进行分析。结果成功扩增出了23条HBV基因组序列，向NCBI提交的24株HBV全长序列，测序结果分析显示，B基因型11例，C基因型12例，没有B＋C型，发现YMDD耐药株1例。结论In．Fusion技术构建重组质粒是一种高效的连接方法，提高了连接率与成功率，可以大大简化目的片段与载体的连接，有效避免了在连接过程中常见的载体自连现象，探索了该技术在HBV序列研究中的初步应用。本研究丰富了我国的HBV全长基因组数据库，为HBV基因组特点、变异、药物、疫苗研究以及乙型肝炎的个体化治疗提供了参考依据。

Objective To investigate the use of In-Fusion technology in the sequencing of hepatitis B virus genome and analyzing the sequence of 23 strains of isolates. Methods Twenty-three strains of type B HBV genome were amplified by PCR from the serum collected in Zhejiang, China, and were inserted into the vector of pMD18T in the ways of In-Fusion technology, and the sequence of it was analyzed. Results The strains of the HBV isolates were types B and C, which were most common in China, and one isolate of YMDD mutation was found. Conclusion It was proven that the In-Fusion technology is an effective way to construct recombinant plasmid, avoiding self-connection of vector. The finding of this research provides a reference to the study of the genotype and evolution of hepatitis B virus in China.