父婴传播的乙型肝炎病毒C基因变异研究

目的 探讨乙型肝炎病毒(HBV)父婴传播的可能性。方法 选择父亲为HBV携带者而母亲无任何HBV感染标志的新生儿为研究对象,检测新生儿的HBV感染标志,比较父亲与子代所携HBV C基因nt2022～2301段序列同源性。结果 16对父亲与新生儿所携HBVC基因同源性在99％～100％,检出2029、2034、2044、2059、2078、2095、2104、2154、2161、2169、2189、2201、2233、2251、2284、2288、2293位核苷酸变异,其中2189、2288位核苷酸变异为表型变异。2189位点A变C致使C区第97位氨基酸由异亮氨酸变为亮氨酸,2288位点C变A使C区第130位氨基酸由脯氨酸变为苏氨酸,其他位点为无表型变异。6对(37．5％)父亲与新生儿存在上述表型变异。结论 存在HBV的父要传播途径且存在主要变异株。

HBV C gene mutation in the transmission from father to infant

OBJECTIVE: Hepatitis B virus (HBV) DNA was detected from infants whose mothers were negative for all HBV markers and the fathers were HBV carrier, the homology of HBV sequence of fathers and fetus was high, and HBV mutations concentrated on some points, and the transmission of HBV from father to fetus was also identified in some reports. The present study aimed to study HBV transmission from father to infant. METHODS: The study enrolled 16 pairs of fathers who were HBV carriers and infants whose mothers were negative for HBV markers. The infants had evidences for intrauterine HBV infection. The five HBV serum markers HBsAg, HBeAg, anti-HBe, anti-HBs, and anti-HBc were detected with ELISA. The positive results for HBsAg and/or HBeAg were regarded as markers of HBV infection. Amplification of HBV DNA was done using a nested PCR method. The first amplification was carried out using primer C1 (nt 2394-2370), and primer C3 (nt 1730-1754). The second amplification was carried out using primer C2 (nt 1955-1974) and primer C6 (nt 2348-2330). Both primers were designed to amplify the part of sequence coding for the hepatitis B C antigen. The size of the amplified fragment obtained by the nested PCR was expected to be 394 bp. The PCR products were electrophoresed on 1.5% agarose gels, which were then stained with ethidium bromide and observed with ultraviolet transillumination. When 394 bp specific band was detectable, the sample was designated positive. Then the positive samples were identified by dot blot. The second PCR products were extracted by phenol-chloroform and 70% ethanol precipitation, then resuspended in TE buffer (pH8.0), and used as the template for cloning. The template was connected into pGEM-T vector by ligase. The ligated products were cloned into fresh competent JM109 cells, and incubated for 90 minutes at 37 degrees C on roller drum. Finally several dilutions were plated on plates containing ampicillin, X-Gal and IPTG, and incubated at 37 degrees C overnight. The white colony on plates was used for identification by the nested PCR with the above primers. When the 394 bp band was detectable by electrophoresis of PCR products in 1.5% agarose gels, the colony was designated positive; a positive colony was incubated in LB medium for 8 to 12 hrs, then plasmid was extracted using the Wizard Plus SV Minipreps DNA Purification System Kit (Promega). The purified plasmid was sent to Beijing Saibaisheng Company for sequencing. The homology of HBV C nt 2022-2301 sequence was compared between fathers and infants. RESULTS: The homology of HBV C nt 2022-2301 sequence were 99% - 100% in 16 pairs of fathers and infants. The results were referred to the published sequence of HBV adw/adr clones, and the nucleic acid databases were searched for homology by using BLAST tool on Internet. HBV of the sixteen pairs of father/infant was closely related to the Japan strain (Genebank accession number AF121249), but there were still 17 more mutations at nucleotide positions 2029, 2034, 2044, 2059, 2078, 2095, 2104, 2154, 2161, 2169, 2189, 2201, 2233, 2251, 2284, 2288, 2293. Moreover the mutations at positions 2189, 2288 resulted in the substitution of the encoded amino acid (corresponding to amino acid positions 97 and 130, respectively), the other mutations at the position were nonphenotypic. The mutation of 2189, 2288 nucleotide of HBV C gene caused 97, 130 amino acid substitution for isoleucine to leucine and proline to threonine. The mutation of 2189, 2288 nucleotide of HBV C gene were detected in 6 (37.5%) of 16 pairs of fathers and infants. CONCLUSION: The HBV transmission from father to infants did exist. The main HBV C gene mutation strains also existed in the transmission.