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目的:探讨不孕女性中人巨细胞病毒(HCMV)感染流行株的包膜糖蛋白B(gB)基因型分布及其与不孕的相关性。方法:采用酶联免疫吸附试验(ELISA)检测300例女性不孕患者血清中HCMV-IgM抗体,ELISA阳性的患者采集晨尿接种于人胚肺成纤维细胞(HELF),提取细胞病变(CPE)阳性培养液中的病毒DNA,以巢式PCR(nest PCR)法扩增HCMVgB基因,利用限制性核酸内切酶HinfΙ、RsaΙ对HCMV gB基因进行限制性片段长度多态性(RFLP)分析判断基因型别;随机抽取11例送检测序,测序结果使用Clustal X软件与GenBank标准病毒株进行序列比对,用MEGA4.1构建核苷酸序列的基因树。结果:300例女性不孕患者中HCMV-IgM抗体阳性45例,阳性率为15.0%。45例患者晨尿接种HELF细胞,观察1月后检测到的病毒阳性37例,阳性率为12.3%。37例病毒阳性患者中最常见的基因型为gB 1型25例(67.6%),其次为gB 3型7例(18.9%)和gB 2型5例(13.5%),没有检测到gB4基因型。测序结果与GenBank标准病毒株HCMVADl69和Towne进行序列比对后,用MEGA4.1成功构建基因树。结论:女性不孕症的发生与HCMV感染有明显相关性,HCMV感染导致的不孕中最常见的gB基因型为gB 1型,其次是gB 3、gB 2型,未检测到gB 4型。
Objective: To investigate the distribution of glycoprotein B (gB) genotypes in infective women infected with human cytomegalovirus (HCMV) and its relationship with infertility. Methods: Serum HCMV-IgM antibody was detected by enzyme-linked immunosorbent assay (ELISA) in 300 female infertile patients. Morning urine samples were taken from human embryo lung fibroblasts (HELF) to collect cytopathic effect (CPE) Positive medium, HCMVgB gene was amplified by nest PCR, and restriction fragment length polymorphism (RFLP) analysis was used to determine the HCMV gB gene by using restriction endonucleases HinfI and RsaI The genotypes were randomly selected from 11 cases and sequenced. The sequencing results were compared with GenBank standard strains using Clustal X software, and the gene sequence of the nucleotide sequence was constructed with MEGA4.1. Results: There were 45 cases of HCMV-IgM positive in 300 cases of female infertility, the positive rate was 15.0%. Forty-five patients were inoculated with morning urine to infect HELF cells, and 37 positive cases were detected after January, the positive rate was 12.3%. Among the 37 virus-positive patients, the most common genotypes were gB1 genotype in 25 cases (67.6%), followed by gB3 genotype in 7 cases (18.9%) and gB2 genotype in 5 cases (13.5%), with no genotype gB4 detected . After sequence alignment with GenBank standard strains HCMVADl69 and Towne, the gene tree was successfully constructed with MEGA4.1. Conclusion: The incidence of infertility in women is significantly correlated with HCMV infection. The most common gB genotype in infertility caused by HCMV infection is gB type 1, followed by gB type 3 and gB type 2, while gB type 4 is not detected.