N-端融合蛋白对重组HBeAg抗原性的影响

目的　研究 Ｎ－ 末端融合蛋白对大肠杆菌中表达的 Ｈ Ｂｅ Ａｇ 抗原性的影响。方法　用 Ｐ Ｃ Ｒ 法，从抗－ Ｈ Ｂｃ（ ＋ ） 血清标本中获得 Ｈ Ｂ Ｖ Ｐｒｅ ｃ － ｃ 基因片段，将其插入质粒 Ｔｒｃ９９ Ａ，重组成亚克隆 Ｐ Ｈ Ｂ Ｉ。以 Ｐ Ｈ Ｂ Ｉ为模板，扩增获得编码乙肝病毒核心蛋白１ ～１４０ 氨基酸的基因片段，加上终止信号，分别插入质粒ｐ Ｇ Ｅ Ｘ－ ２ Ｔ 和 Ｔｒｃ９９ Ａ 中，各自转化后，以 Ｉ Ｐ Ｔ Ｇ 诱导，大肠杆菌表达插入基因。以 Ｅ Ｌ Ｉ Ｓ Ａ 和 Ｓ Ｄ Ｓ－ Ｐ Ａ Ｇ Ｅ 方法测定表达产物的分子量和 Ｈ Ｂｅ Ａｇ 、 Ｈ Ｂｃ Ａｇ 滴度。结果　在 Ｉ Ｐ Ｔ Ｇ 诱导下，大肠杆菌中分别表达 Ｎ－ 端融合的 Ｇ Ｓ Ｔ－ Ｈ Ｂｅ Ａｇ 融合蛋白和非融合 Ｈ Ｂｅ Ａｇ 蛋白。分子量各为４１０００ Ｄ 和１５０００ Ｄ。培养裂解物经 Ｅ Ｌ Ｉ Ｓ Ａ 测定，融合蛋白 Ｈ Ｂｅ Ａｇ ： Ｈ Ｂｃ Ａｇ 的滴度比为２５６ ：１ ，非融合蛋白为１６ ：１ 。结论　 Ｎ－ 端融合蛋白 Ｇ Ｓ Ｔ－ Ｈ Ｂｅ Ａｇ 抗原性更接近血清 Ｈ Ｂｅ Ａｇ 。

Changes in antigenicity of HBeAg fusing N-protein expressed in E.coli

Objective To analyse changes in antigenicity of HBeAg fusing N-protein expressed in E.coli. Methods By using PCR,HBV Pre c～c gene was obtained from Anti-HBc positive serum sample. The gene was inserted to plasmid Trc 99A and recombinated to subclon P HBI I.The gene fragment coding HBV core protein's 1～140 amino acids was amplyfied from P HBI I.Stop code were added to the gene fragments and inserted to plasmid pGEX-2T and Trc 99A.The transformants were induced by IPTG and expressed inserted gene in E.coli. ELISA and SDS-PAGE were used to assay two kinds of lytic culture. Results The transformants express HBeAg fused GST at amino terminus or unfusion HBeAg in E.coli.GST-HBeAg Mw:41000D,unfusion HBeAg Mw:15000D. The titer ratio HBeAg:HBcAg of fusion protein was 256:1,the ratio in unfusion protein was 16:1. Conclusion The HBeAg fused N-protein is more similar to serum HBeAg in e antigenicity.