乙型肝炎病毒表面抗原aa126 Ile→Ser变异株HBsAg的暂时表达和抗原性鉴定

TRANSIENT EXPRESSION AND ANTIGENIC CHARACTERIZATION OF HBsAg126Ser OF HEPATITIS B VIRUS aa126Ile→Ser MUTANT

曾在一个儿童患者体内，发现一个新的乙型肝炎病毒（ＨＢＶ）变异株，其ＨＢｓＡｇ主蛋白ａａ１２６发生Ｉｌｅ（ＡＴＴ）到Ｓｅｒ（ＡＧＴ）的取代。已知ａｄｒ／ａｙｒ亚型ＨＢｓＡｇ１２６位为Ｉｌｅ，而ａｄｗ／ａｙｗ亚型ＨＢｓＡｇ１２６位为Ｔｈｒ，表明ＨＢｓＡｇ１２６Ｓｅｒ是一个新的变异株。用计算机做结构分析的结果表明，突变体ＨＢｓＡｇ１２６Ｓｅｒ主蛋白ａａ１２０－ａａ１３０区段的二级结构与野生型ａｄｒＨＢＶＨＢｓＡｇ１２６Ｉｌｅ相比发生明显的改变。这种构象的变化可能会影响ａ抗原决定簇（ａ１２４－ａａ１４７）的抗原性。为了证实这一点，构建了突变Ｓ基因表达质粒，在ＳＶ４０早期启动子的控制下进行抗原表达。利用ＨＢｓＡｇ９肽（Ｔｈｒ－Ｉｌｅ１２６－Ｐｒｏ－Ａｌａ－Ｇｌｎ－Ｇｌｙ－Ｔｈｒ－Ｓｅｒ－Ｍｅｔ）抗ｉ单克隆抗体和９肽（Ｔｈｒ－Ｔｈｒ１２６－Ｐｒｏ－Ａｌａ－Ｇｌｎ－Ｇｌｙ－Ｔｈｒ－Ｓｅｒ－Ｍｅｔ）抗ｔ单克隆抗体进行放射免疫测定，结果表明，这种Ｓｅｒ１２６突变蛋白对抗ｉ单克隆抗体的反应性比野生蛋白减弱，对抗ｔ单克隆抗体的反应性则与ＨＢｓＡｇ１２６Ｔｈｒ接近。但用三种抗－ａ单克隆抗体的检测结果揭示，突变蛋白ＨＢｓＡｇ１２６?

A novel hepatitis B virus(HBV)mutant from the serum of a child carrier in Shanghai was characterized. The child had been immunized with HBV vaccine, but failed to produce antibody against hepatitis B surface antigen(HBsAg). The serum was positive for HBsAg and had a high level of alanine aminotransferase(ALT). The complete nucleotide sequence of HBsAg major protein coding region showed that this HBV was a subtype adr with four nucleotide changes and one of them was located within the coding region of a determinant of HBsAg. T at nt 531 of HBV genome was replaced by G, leading to an amino acid substitution of isoleucine to serine at residue 126 of HBsAg(Fang D, et al. Acta Biochem. Biophys. Sinica, 1996, 28(4): 429-433). It is known that the residue 126 of HBsAg major protein is Ile for adr/ayr subtype and Thr for adw/ayw subtype. The HBsAg126Ile→Ser is a new variant worth to study in more detail. Computer analysis of the polypeptide deduced from the HBsAg major protein coding sequence showed that the secondary structure in the region between aa120 and aa130 of the HBsAg126Ser changed significantly, as compared with the known adr subtype. This conformational change should alter the antigenicity of the a determinant(aa124 to aa147). To verify this, expression plasmid containing the mutant S gene was constructed and expressed in COS-M6 cells under the regulation of SV40 early promoter. Radioimmunoassay analysis with an anti-i monoclonal antibody specific for an enneapeptide(Thr-Ile126-Pro-Ala-Gln-Gly-Thr-Ser-Met)showed that the binding activity of HBsAg126Ser was much weaker than the wild HBsAg major protein, but gave a higher level of binding to an anti-t monoclonal antibody specific to the enneapeptide with Thr126. RIA with three different anti-a monoclonal antibodies showed that the binding activity of the mutant HBsAg126Ser was stronger than that of the wild counterpart. It seems that the aa126Ser mutant of HBV is a new mutant different from the previously reported vaccine induced escape mutant.