HBV亚基因型B和C体外重组中间体的检测

摘要

目的:检测HBV亚基因型B和C的体外重组中间体.方法:将基因型B和C序列插入载体Plenti6/V5-D-topo-X后,共转染HepG2细胞,克隆,测定转染后HBV的核酸序列,而后用软件包RDP3Beta40进行序列比对.结果:发现存在3种重组中间体,重组中间体的重组位置为1740-1838至2443-2485.R1重组中间体的重组位置为nt2170-2172(CAC变成TGT)和 nt2188-2189(GA变成AC);R2重组中间体的重组位置为nt1740(A变成 T), nt1753(C变成 T )和 nt1838 (G 变成 A);R3重组中间体的重组位置为nt2443(C 变成 T), nt2425 (A 变成 G), nt2480( T变成 C)和nt2483 (C 变成T).结论:HBV基因型B和C共感染可以产生重组,且重组位置在基因型B和C的前核心区和核心区.%Objective To detect the recombinant intermediates of hepatitis B virus (HBV) between genotype B and C in vitro.Methods Vector Plenti6/V5-D-topo-X was genetically modified by genotype B and C to transfect HepG2 cells.Then the HepG2 cells were amplified and sequence of the nucleic acid after the transinfection was tested and compared with RDP3 Beta40 software package and bootscanning procedure in SimPlot program package.Results Three recombinant intermediates of HBV between genotype B and C were identified in vitro.Genotype C in the precore region plus the core gene spanning nucleotide positions from 1 740 - 1 838 to 2 443 - 2 485 contributed to the recombination with genotype B.Isolate R1 recombinant intermediate had 2 break points at nt 2 170 -2 172 and nt2 188 -2 189.Nucleic acid changed from CAC to TGT and from GA to AC,respectively.Isolate R2 recombinant intermediate had a break point at nt 1 740 - 1 838, and 3 bases changed in different nucleic acid sites: from A to T at nt 1 740, from C to T at nt 1 753, and from G to A at nt 1 838, respectively.Isolate R3 recombinant intermediate had a break point at nt 2 4432 483, and 4 bases changed in different nucleic acid sites: from C to T at nt 2 443, from A to G at nt 2 452, from T to C at nt 2 480, and from C to T at nt 2 483, respectively.Conclusion The recombinant intermediates of HBV between genotype B and C have been detected in vitro and the changes have been identified in the precore region plus the core gene in genotype B and C.