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目的分析中国部分甲型肝炎(甲肝)病毒(Hepatitis A Virus,HAV)流行株结构蛋白VP3~VP1编码区核苷酸及氨基酸序列特点,并比较与甲肝减毒活疫苗(Hepatitis A Attenuated Live Vaccine,Hep A-L)标本序列差别。方法收集39份甲肝病例急性期血清样本及2份市售Hep A-L标本,经核酸提取、逆转录及巢式聚合酶链反应,测得结构-非结构蛋白VP3~VP1-2A编码区序列,进行基因亲缘关系、氨基酸序列同源性等分析。结果检测到的部分HAV流行株序列均为IA亚型,其在结构-非结构蛋白VP3~VP1-2A编码区核苷酸和氨基酸序列同源性分别为94.8%~100%和99.3%~100%,与检测的2份Hep A-L标本的核苷酸和氨基酸序列同源性为90.6%~91.9%和99.1%~99.4%。其中的1条序列在VP3-56位,发生丝氨酸(Serine,Ser)→脯氨酸(Proline,Pro)替代;3条序列在VP1-112位,发生苏氨酸(Threonine,Thr)→异亮氨酸(Isoleucine,Ile)替代,靠近中和抗原位点VP1-114位;所有序列在已发表的中和抗原位点处氨基酸无变化。选择压力分析表明,检测到的部分HAV流行株序列在VP3~VP1编码区处于负向选择。检测的2份Hep A-L标本序列均为IB亚型,在已发表的中和抗原位点处氨基酸无变化。结论HAV的结构蛋白编码区核苷酸序列存在一定差异,但其氨基酸序列相对保守。检测到的3条HAV流行株序列在中和抗原位点附近发生氨基酸替代,值得进一步研究。
Objective To analyze the nucleotide and amino acid sequences of the coding region of the VP3 ~ VP1 structural protein in some Chinese HAV strains and to compare their nucleotide and amino acid sequences with those of the live attenuated hepatitis A vaccine (Hepatitis A Attenuated Live Vaccine, Hep AL) specimen sequence differences. Methods Serum samples from 39 patients with acute hepatitis A and 2 commercially available samples of Hep AL were collected and sequenced by nucleic acid extraction, reverse transcription and nested polymerase chain reaction (PCR-RFLP) Genetic relationship, amino acid sequence homology analysis. Results The sequences of some HAV strains detected were all subtype IA. The nucleotide and amino acid sequence homologies of the structurally non-structural proteins VP3 ~ VP1-2A were 94.8% -100% and 99.3% -100 %. The nucleotide and amino acid sequence identities of the two Hep AL samples tested ranged from 90.6% to 91.9% and from 99.1% to 99.4%. One of these sequences was replaced by serine (Ser) → Proline (Pro) at VP3-56. Threonine (Thr) → isoleucine Isoleucine (Ile), near the site of neutralizing antigen VP1-114; all sequences showed no change in amino acid at the published neutralization antigenic site. Selective pressure analysis showed that some of the detected sequences of HAV strains were negatively-selected in the VP3-VP1 coding region. The two Hep A-L samples tested were all of subtype IB, with no change in amino acid at the published neutralizing antigen site. Conclusion There are some differences in the nucleotide sequence of the structural protein coding region of HAV, but its amino acid sequence is relatively conservative. The detected sequences of three HAV strains were amino acid substitution near the neutralizing antigen site, which deserved further study.