病毒性肝炎 Open Access
Copyright ©The Author(s) 2003. Published by Baishideng Publishing Group Inc. All rights reserved.
世界华人消化杂志. 2003-07-15; 11(7): 966-969
在线出版日期: 2003-07-15. doi: 10.11569/wcjd.v11.i7.966
乙型肝炎病毒核壳蛋白变异株在HepG2细胞的HLA- I表达
陈伟红, 何海棠, 张明霞, 刘志华, 周永兴
陈伟红, 周永兴, 中国人民解放军第四军医大学唐都医院传染科陕西省西安市 710038
陈伟红, 女, 1964-10-13生, 浙江省嘉兴市人, 汉族, 医学博士生, 讲师.
何海棠, 张明霞, 刘志华, 中国人民解放军第一军医大学南方医院传染科广东省广州市 510515
通讯作者: 陈伟红, 710038, 陕西省西安市, 中国人民解放军第四军医大学唐都医院传染科. chenwhong101@163.com
电话: 029-3377752 传真: 029-3537377
收稿日期: 2002-10-09
修回日期: 2002-10-20
接受日期: 2002-11-09
在线出版日期: 2003-07-15

目的

研究HBV adr亚型野生株和核壳蛋白变异株在HepG2细胞表面的HLA-I/抗原肽复合物的表达.

方法

通过定点突变技术将1.2拷贝HBV野生型质粒p3.8Ⅱ构建成核壳蛋白变异株V60和L97. 经序列测定和生物学活性检测后, 野生株和变异株重组质粒分别亚克隆入EB病毒表达载体EBO-plpp以稳定表达. 重组载体EBO-野生株、EBO-V60和EBO-L97分别作内切酶双酶切和序列测定鉴定, 再用脂质体介导转染HepG2细胞, ELISA(Abbott)试剂盒定量检测各株培养上清HBV抗原, 转染细胞用FITC标记的鼠抗HLA-ABC单抗染色, 流式细胞术分析细胞表面HLA-I表达.

结果

3株重组载体经内切酶消化, 电泳后显示2条区带, 分别与1.2拷贝HBV基因组和EBO载体大小相同. 测序结果证实EBO-V60和EBO-L97分别在nt2078 C→G和nt2189 A→C, 保持原定点突变. EBO-野生株的培养上清HBeAg定量S/CO值明显高于变异株V60和L97, 3株HBsAg定量S/N值接近, HBsAg表达相近表明实验的转染效率相当. EBO空载体转染的HepG2细胞HLA-I轻微表达, 3株重组载体转染细胞HLA-I的荧光强度不同, 野生株增强为18.2, L97明显升高至34.5, 而V60降低至3.4.

结论

HBV能增强HepG2 细胞表面HLA-I/抗原肽复合物的表达, 核壳蛋白热点变异V60和L97可使宿主细胞HLA-I表达发生变化.

关键词: N/A
引文著录: 陈伟红, 何海棠, 张明霞, 刘志华, 周永兴. 乙型肝炎病毒核壳蛋白变异株在HepG2细胞的HLA- I表达. 世界华人消化杂志 2003; 11(7): 966-969
Expression of HLA-I on HepG2 cells by hepatitis B virus nucleocapsid mutants
Wei-Hong Cheng, Hai-Tang He, Ming-Xia Zhang, Zhi-Hua Liu, Yong-Xing Zhou
Wei-Hong Cheng, Yong-Xing Zhou, Department of Infectious Diseases, Tangdu Hospital, the Fourth Military Medical University, Xi'an 710038, Shaanxi Province, China.
Hai-Tang He, Ming-Xia Zhang, Zhi-Hua Liu, Department of Infectious Diseases, Nanfang Hospital, the First Military Medical University, Guangzhou 510515, Guangdong Province, China.
Correspondence to: Wei-Hong Cheng, Department of Infectious Diseases, Tangdu Hospital, the Fourth Military Medical University, Xi'an 710038, Shaanxi Province, China. chenwhong101@163.com
Received: October 9, 2002
Revised: October 20, 2002
Accepted: November 9, 2002
Published online: July 15, 2003

AIM

To study the expression of HLA-I/antigen peptide complex on HepG2 cells transfected with HBV (adr) wild type and nucleocapsid protein mutants.

METHODS

The site-directed mutation was performed to introduce nucleocapsid protein point mutations V60 and L97 into 1.2 copies of HBV genome plasmid p3.8 Ⅱ. After identification of DNA sequence and biological activities, the plasmid p3.8Ⅱ and mutant plasmid constructs were subcloned respectively into EB virus based vector EBO-plpp for stable expression. The vector constructed EBO-wild type, EBO-V60, and EBO-L97 were analyzed by restriction enzyme digestion and DNA sequenceing, then transfected into HepG2 cells via the liposome technique, respectively. HBV antigen in their culture supernatants was quantified by Abbott kits. The cells were stained with murine monoclonal antibody anti-HLA-ABC conjugated directly to FITC, and expression of HLA-I on their membrane was analyzed by flow-cytometry.

RESULTS

Restriction enzyme digestion of 3 vector constructs showed two bands similar to HBV 1.2 copies genome and EBO vector, respectively. Analysis of DNA sequence confirmed the mutated nucleotides of EBO-V60 and EBO-L97 (i.e nt2078 C→G, nt2189 A→C). The expression of HBeAg S/CO in culture supernatant of EBO-wild type was much higher than that of mutant EBO-V60 and EBO-L97, while the expression of HBsAg S/N in three constructs had similar level, indicating similar transfecting rate in this experiment. The expression of HLA-I on HepG2 cells transfected with EBO empty vector was at low level. Fluorescence intensity of HLA-I expression of transfected cells was elevated by EBO-wild type (18.2), while that of L97 was increased to 34.5 and V60 declined to 3.4.

CONCLUSION

HBV might enhance the expression of HLA-I/antigen peptide complex on HepG2 cells. Hot-spot mutations of HBV nucleocapsid protein L97 and V60 could influence the expression level of HLA-I on host cells.

Key Words: N/A
0 引言

细胞表面的人类白细胞抗原(human leukocyte antigen, HLA)是激发并调节机体T细胞免疫应答的关键分子, HLA与HBV的感染、病毒诱导的免疫应答和免疫病理的相关性已受到广泛关注[1-9]. HBV C基因编码的核壳蛋白是免疫应答的主要靶抗原, 免疫效应细胞CTL通过TCR识别并结合HLA-I/病毒肽复合物实施细胞毒作用. 慢性感染者的HBV C基因长期处于免疫选择压力下常发生聚集性变异, 其中AA97的点突变占错义变异45%以上[10-12]. 我国HBV主要流行亚型adr型的Ｃ基因变异中以AA97的异亮氨酸ATC→亮氨酸CTC(L97)为最常见, AA60亮氨酸CTG→缬氨酸GTG(V60)为另一热点变异. 我们构建HBV(adr)全基因野生株及L97, V60变异株EBO真核表达载体, 体外实验分析各株诱导HepG2细胞表面HLA-I/抗原肽复合物表达的特性, 探讨核壳蛋白热点变异对HLA-I表达的影响.

1 材料和方法
1.1 材料

含1.2拷贝HBV adr亚型基因组质粒p3.8Ⅱ由中国科学院生物化学研究所汪垣教授惠赠, 为野生株3.2 kb基因组3'端重叠587 bp(nt1402-1987)片段插入pBS+的重组质粒. EB病毒稳定真核表达载体EBO-plpp由美国Scripps研究所Francis V. Chisari博士惠赠. 根据Gan et al (Zhongguo Kexue B Gi 1986; 1: 55-65)报道的我国HBV adr核苷酸序列设计PCR引物(表1).

表1 PCR引物核苷酸序列.
引物序列(5'→3')
L97点突变引物 L1GGCCTAAAACTCAGACAACTA
L2TAGTTGTCTGAGTTTTAGGCC
V60点突变引物 V1AGGCAAGCTATTGTGTGTTGGGGTG
V2CACCCCAACACACAATAGCTTGCCT
测序引物 Pee2GCGGGTACCCTAACATTGAGATTCCCG
亚克隆引物 P1GCTGTCGACCGCTTGATGCCT
Sal Ⅰ
P2CCTTCTCGGTACCGGGGATCTA
Kpn Ⅰ
1.2 方法

p3.8Ⅱ变异株重组质粒的构建采用Quik ChangTM定点突变试剂盒(Stratagene), 以质粒p3.8ⅡDNA为模板, 分别加入L97及V60点突变引物L1, L2及V1, V2进行定点突变, 构建成p3.8ⅡL97和p3.8ⅡV60重组质粒. 变异株重组质粒用Pee2引物扩增后进行全自动测序(上海基康生物公司). 将p3.8Ⅱ野生株(WT)及变异株重组质粒以脂质体(Gibco)介导分别转染HepG2细胞, 用含100 mL/L胎牛血清的DMEM培养液(Gibco)培养96 h, 收集细胞和培养上清, 用Southern印迹杂交(HBV地高辛标记探针)分析细胞内HBV DNA的复制, ELISA法测定上清中HBV抗原, 以检测其生物活性. EBO-野生株及变异株重组载体的亚克隆构建以p3.8ⅡWT, p3.8ⅡL97及p3.8ⅡV60质粒DNA为模板, 各自加入5'端带Sal I及Kpn I酶切位点的引物P1及P2, 用Z-Taq DNA聚合酶(TaKaRa)进行PCR扩增, 各株PCR产物及EBO-plpp载体分别用Sal I 及Kpn I(Promega)双酶切, 电泳后凝胶回收, 用T4DNA连接酶(Promega)在16 ℃连接过夜, 转化感受态细菌XL-1, 筛选阳性克隆. 各株重组载体阳性克隆用Sal I 及Kpn I 双酶切, 进行凝胶电泳. 以Pee2引物PCR扩增各株阳性克隆, 再次作自动化测序(上海基康生物公司). 将EBO-WT, EBO-L97, EBO-V60重组载体及EBO空载体DNA各2 μg, 经脂质体10 μL介导转染HepG2细胞, 用潮霉素200 mg/L筛选, 每2 d定量更换培养液, 建立各株稳定表达的细胞. 转染实验重复进行3次. 用ELISA(Abbott)试剂盒测定各株细胞培养上清中HBsAg和HBeAg的含量, 分别以S/N值和S/CO值表示. 收集各株转染的HepG2细胞2×106个, 以FITC标记的鼠抗HLA-ABC单克隆抗体(Phar Mingen)室温避光染色30 min, PBS洗2次, 40 mL/L多聚甲醛固定, 以未转染的HepG2细胞作阴性对照, 用流式细胞仪检测, 分析5104个细胞的荧光强度.

2 结果
2.1 p3.8Ⅱ变异株重组质粒序列测定及生物活性检测

变异株质粒DNA测序结果, p3.8ⅡL97的nt2189 A→C, p3.8ⅡV60的nt 2 078 C→G, 证实定点突变. p3.8Ⅱ变异株及p3.8Ⅱ野生株转染细胞的Southern印迹杂交显示在3.8 kb处呈现一明显杂交带, 符合目的基因组大小, 表明所构建的重组质粒均能在细胞内复制, 细胞培养上清中均测出HBsAg和HBeAg, 有HBV抗原表达.

2.2 EBO重组载体的酶切鉴定及序列测定

所构建的EBO-WT, EBO-97及EBO-V60重组载体DNA各经Sal I 及Kpn I内切酶双酶切, 电泳后均出现二条区带, 分别与HBV目的基因组及EBO表达载体的大小相符. EBO-L97及EBO-V60 DNA测序结果显示均保持原定的点突变.

2.3 EBO重组载体转染细胞培养上清HBsAg和HBeAg定量检测

3次转染实验的细胞培养上清中HBV抗原平均含量, 3株表达的HBeAg S/CO值不同, EBO-L97及EBO-V60均明显低于EBO-WT, 3株上清的HBsAg S/N值接近, 表达水平相似(表2).

表2 转染细胞培养上清HBV抗原含量.
HBV抗原EBO-WTEBO-L97EBO-V60
HBsAg(S/N)4.343.964.18
HBeAg(S/CO)3.300.981. 52
HBsAg含量S/N参考值<2.00 HBeAg含量S/CO参考值<1.00.
2.4 宿主细胞表面HLA-I表达

流式细胞术分析测定3批转染实验的HepG2细胞. 未转染的及EBO空载体转染的HepG2细胞表面的HLA-I为轻微表达, 其荧光强度为1.8和2.2. 3株重组载体转染细胞的HLA-I表达水平各不相同, 野生株的HLA-I荧光强度增强为18.2, L97变异株升高至34.5, 而V60变异株表达的荧光强度仅为3.4(图1).

图1
图1 流式细胞仪对HepG2细胞表面HLA-I表达的检测. A: 空白未转染; B: EBO空载体转染; C: EBO-WT转染; D: EBO-L97转染; E: EBO-V60转染.
3 讨论

HBV并非直接导致肝细胞病变, 而是其编码蛋白与机体免疫应答相互作用后, 引起肝组织发生免疫病理损伤, 同时抑制和清除细胞内外病毒, 在免疫应答过程中HLA-I类分子限制的特异性CD8+T细胞的细胞毒作用十分重要[13-18]. 正常肝细胞表面不表达或仅微量表达HLA-I类分子. 研究已表明HBV感染者肝细胞膜上HLA-I表达强度与肝组织病变活动性及损伤程度往往呈正相关, 急性重型肝炎患者为强表达, 慢性感染者在免疫耐受期为弱表达, 免疫激活期HLA-I表达增强, 肝组织炎症活动, 部分病毒被清除[4,6,19]. 机体内细胞膜上HLA-I的表达受到病毒及免疫细胞激活后产生的细胞因子及其他诸多方面的诱导和调节[20,21], 建立细胞模型可探讨病毒本身与宿主细胞间的相互作用. 由于HBV基因组结构复杂, 编码基因相互重叠, 基因内部有调节序列[10,22,23], 单一基因片段的体外表达有局限性, 用全基因重组质粒转染细胞更接近病毒在体内的复制、表达、加工和分泌情况. 我们用HBV adr亚型野生株含1.2拷贝基因组质粒p3.8Ⅱ, 对核壳蛋白热点变异97位氨基酸及60位氨基酸分别进行定点突变, 再双酶切定向亚克隆入EB病毒真核表达载体EBO-plpp, 能在转染的HepG2细胞中稳定表达, HepG2细胞的HLA抗原型别以HLA-A2为主, HLA-A2在亚洲人群中携带频率最高, 因而本实验采用了较合理的细胞模型[24-26], 进行核壳蛋白密码子点突变的HLA-I表达的研究. 实验结果显示, HBV野生株重组载体EBO-WT较EBO-plpp空白载体转染和未转染的HepG2细胞表面HLA-I表达明显增强, 与国外(Zhou et al 1990, Takehara et al 1992)体外表达的研究结果相同, 初步表明HBV病毒自身可增强细胞膜上HLA-I表达. 其他病毒可上调感染细胞HLA-I表达的实验研究报道也不少, 如黄热病病毒家族、呼吸道合胞病毒、单纯疱疹病毒、人副流感病毒3型等[27-31].

EBO-WT, EBO-L97及EBO-V60重组载体转染HepG2细胞稳定传代后, 培养上清中HBV抗原定量检测结果, 3株表达HBeAg的水平不同, L97及V60变异株的HBeAg S/CO值明显较野生株为低, 3株的HBsAg S/N值接近, HBsAg表达水平相近, 表明重组载体转染率及检测结果相当, 因而其转染细胞膜上HLA-I 表达分析有一定可比性. 流式细胞术分析3株宿主细胞HLA-I表达, L97变异株HLA-I荧光强度高于野生株, 而V60变异株表达强度低于野生株, 表明HBV核壳蛋白单个错义点突变可使宿主细胞表面HLA-I表达发生差异. Yuan et al[32-34]用HBV adr亚型双拷贝基因组的核壳蛋白变异株做体外实验, 发现L97(I97L)及V60(L60V)变异在HepG2细胞内的复制水平与野生株相近, 但L97分泌未成熟的(含ssDNA)Dane颗粒, 且颗粒数量明显增加, V60则分泌成熟的Dane颗粒, 其量下降3-6倍, 充分说明HBV核壳蛋白上述密码子错义突变能引起病毒的表达、加工、分泌等生物学功能的变化. 我国HBV感染率高[35-40], 关于HBV转染细胞中内源性细胞因子对HLA－I表达的影响以及核壳蛋白L97及V60变异导致宿主细胞HLA-I表达变化的机制, 均有待深入研究探讨.

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