慢性乙型肝炎患者体内乙型肝炎病毒准种特点的初步研究

目的 探讨乙型肝炎病毒（HBV）准种在慢性乙型肝炎患者中的存在状态。方法 以已知中国株HBV基因序列为依据。设计特异性聚合酶链反应（PCR）引物，自3例慢性乙型肝炎患者体内扩增HBV全S基因片段，克隆人pGEMTeasy质粒，用限制性片段长度多态性（RFLP）法确定HBV的变异现象，DNA测序确定病毒的变异程度。结果 分别自3例患者血清中克隆出29、28和8个阳性克隆，以EcoRⅠ酶切患者1和2的RFLP结果提示各有5种不同的长度带型，PCR产物XhoI酶切RFLP结果表现为高度保守，测序结果发现HBV体内毒株DNA序列高度一致，同一患者两株全S区序列测序结果表明DNA序列的同源性大于97%。结论 慢性乙型肝炎患者体内有HBV准种共存，且呈现出一定的优势克隆现象。可能与患者预后有关。

The preliminary study on hepatitis B virus (HBV) quasispecies in patients with chronic HBV infection

Objective To investigate the HBV genome status in the sera of patients with chronic HBV infection. Methods A set of specific primers were designed and synthesized according to HBV DNA sequence. The whole S gene of HBV was amplified by polymerase chain reaction (PCR) technique from the sera of 3 patients with chronic HBV infection and cloned into pGEM Teasy vector. Restriction fragment length polymorphism (RFLP) method with EcoR I digestion of the recombinant plasmids and Xho I digestion of the PCR products to display the mutated status and sequences discrepancy of HBV genomes. The whole S DNA from selec-ted plasmid has been sequenced. Results 1.4 kb HBV DNA fragment including pre-S1, pre-S2 and S regions was amplified by PCR and cloned into pGEM Teasy vector. The 29 clones of HBV S DNA plasmid from patient 1, 28 clones from patient 2 and 8 clones from patient 3 have been identified. RFLP analysis showed that there were at least 5 different kinds of restriction fragment length in each recombinant plasmid group from the patient 1 and 2, and an identical physical map in patient 3. The phy-sical map of PCR products has shown high identity in both groups. Sequencing results proved the high consistency, with more than 97% homology between 2 strains from the same patient. Conclusions There are HBV quasispecies populations in the patients with chronic HBV infection, where preponderant strain can be found in the sera of patients with chronic HBV infection.