乙型肝炎病毒表面抗原中蛋白去糖基化对体液免疫应答的影响

The effect on humoral immunity induced by middle hepatitis B virus surface antigen deglyco-sylation in BALB/c mice

目的:观察乙型肝炎病毒(HBV)表面抗原中蛋白(MHBs)中去糖基化对体液免疫应答的影响。方法:在MHBs核酸疫苗(pSW3891/MHBs/adr)基础上,通过PCR扩增基因修饰法,使MHBs上编码第4、59、146位氨基酸密码子基因由AAT/AAC突变为CAG,从而使AAT/AAC编码的天冬酰胺Asn(N)突变为CAG编码的谷氨酰胺Gln(Q),使得N-糖基化位点NXS、NXT突变为QXS、QXT。构建3株去糖基化核酸疫苗(dG1,去除Asn4处糖基化位点,位于preS2上;dG23,去除Asn59、Asn146处糖基化位点,均位于HBs上;dG123,去除Asn4、Asn59及Asn146位糖基化位点)。脂质体法转染293T细胞,Western blot检测,观察它们在293T细胞中瞬时表达的差异;肌肉注射法免疫BALB/c小鼠,观察MHBs去糖基化对小鼠体液免疫应答的影响。结果:adr、dG23转染293T细胞后,在细胞内及上清液中均检测到HBsAg;dG1、dG123转染293T细胞后,在细胞内检测到HBsAg,但是上清液中未测到。adr、dG1及dG23免疫BALB/c小鼠后均产生抗-HBs抗体,最高滴度均达到1:102 400;dG123几乎无抗体反应(<1∶200)。结论:Asn4去糖基化导致MHBs向细胞外的分泌障碍,但是对小鼠抗-HBs的产生无影响。Asn59、Asn146两处同时去糖基化对MHBs的分泌和小鼠抗-HBs的产生影响轻微。Asn4、Asn59及Asn 146三处同时去糖基化明显影响MHBs的分泌和小鼠抗-HBs的产生。

Objective：To observe the effect on humoral immunity induced by deglycosylated middle hepatitis B virus surface antigen （MHBs） in BALB/c mice. Methods：By the use of gene modification using PCR,Asn encoding codon AAT/AAC were replaced by Gln encoding codon CAG, so the N-glycosylation sites Asn-X-Thr/Ser （NXS/T） were replaced by Gln-X-Thr/Ser（QXS/T）. On the base of a DNA vaccine （pSW3891/MHBs/Adr） encoding MHBs, three new N-linked deglycosylation mutations were engineered：dGi ,deglycosylation of Asn 4,located on the preS2; dG23,deglycosylation of Asn 59 and Asn 146,located on the HBs and dGi23, deglycosylation of Asn4, Asn59 and Asn146. In vitro expressions following transient transfetion in 293T cell line were observed. Also the MHBs antibody titers were studied in BALB/c mice by different deglycosylated MHBs DNA vaccine. Results： HBsAg could be detected both in the supernatant and the cell lysate after transient transfection in 293T cell line with adr and dG23 DNA vaccine. It also could be detected in the cell lysate but not in the supernatant with dGi and dG123. Mice immunized with adr, dG1 and dG23 showed strong anti-HBs response（ending point〉i ：102400）, but not with dG123 （ending point〈1 ：200）. Conclusion： The MHBs secretion in 293T cells was significantly influenced by the deglycosylation of Asn 4 in MHBs, but the antibody level induced by deglycosylated vaccine in the mice was not affected. The MHBs secretion and the antibody titers were slightly influenced by the deglycosylation of both Asn59 and Asn146,but they were completely impacted by the deglycosylation of Asn4,Asn59 and Asn146 simutaneously.