Prokaryotical expression of structural and non-structural proteins of hepatitis G virus

AIM: To study the epitope distribution of hepatitis G virus (HGV) and to seek for the potential recombinant antigens for the development of HGV diagnostic reagents. METHODS: Fourteen clones encompassing HGV gene fragments from core to NS3 and NS5 were constructed using prokaryotic expression vector pRSET and (or) pGEX, and expressed in E.coli. Western blotting and ELISA were used to detect the immunoreactivity of these recombinant proteins. RESULTS: One clone with HGV fragment from core to E1 (G1), one from E2 (G31), three from NS3 (G6, G61, G7), one from NS5B (G821) and one chimeric fragment from NS3 and NS5B (G61-821) could be expressed well and showed obvious immunoreactivity by Western blotting. One clone with HGV framment from NS5B (G82) was also well expressed, but could not show immunoreactivity by Western blotting. No obvious expression was found in the other six clones. All the expressed recombinant proteins were in inclusion body form, except the protein G61 which could be expressed in soluble form. Further purified recombinant proteins G1, G31, G61, G821 and G61-821 were detected in indirected ELISA as coating antigen respectively. Only recombinant G1 could still show immunoreactivity, and the other four recombinant proteins failed to react to the HGV antibody positive sera. Western blotting results indicated that the immunoactivity of these four recombinant proteins were lost during purification. CONCLUSION: Core to E1, E2, NS3 and NS5 fragment of HGV contain antigenic epitopes, which could be produced in prokaryotically expressed recombinant proteins. A high-yield recombinant protein (G1) located in HGV core to E1 could remain its epitope after purification, which showed the potential that G1 could be used as a coating antigen to develop an ELISA kit for HGV specific antibody diagnosis.

Full-length core sequence dependent complex-type glycosylation of hepatitis C virus E2 glycoprotein

AIM: To study HCV polyprotein processing is important for the understanding of the natural history of HCV and the design of vaccines against HCV. The purpose of this study is to investigate the affection of context sequences on hepatitis C virus (HCV) E2 processing. METHODS: HCV genes of different lengths were expressed and compared in vaccinia virus/T7 system with homologous patient serum S94 and mouse anti-serum M( E2116) raised against E.coli -derived E2 peptide, respectively.Deglycosylation analysis and GNA ( Galanthus nivalus ) lectin binding assay were performed to study the post-translational processing of the expressed products. RESULTS: E2 glycoproteins with different molecular weights (-75 kDa and -60 kDa) were detected using S94 and M( E2116), respectively. Deglycosylation analysis showed that this difference was mainly due to different glycosylation. Endo H resistance and its failure to bind to GNA lectin demonstrated that the higher molecular weight form (75 kDa) of E2 was complex-type glycosylated, which was readily recognized by homologous patient serum S94. Expression of complex-type glycosylated E2 could not be detected in all of the core-truncated constructs tested, but readily detected in constructs encoding full-length core sequences. CONCLUSION: The upstream conserved full-length core coding sequence was required for the production of E2 glycoproteins carrying complex-type N-glycans which reacted strongly with homologous patient serum and therefore possibly represented more mature forms of E2. As complex-type N-glycans indicated modification by Golgi enzymes, the results suggest that the presence of full-length core might be critical for E1/E2 complex to leave ER. Our data may contribute to a better understanding of the processing of HCV structural proteins as well as HCV morphogenesis.

Acute hepatitis C in a chronically HIV-infected patient:Evolution of different viral genomic regions

AIM: To analyze the molecular evolution of different viral genomic regions of HCV in an acute HCV infected patient chronically infected with HIV through a 42-month follow-up.METHODS: Serum samples of a chronically HIV infected patient that seroconverted to anti HCV antibodies were sequenced, from the event of superinfection through a period of 17 months and in a late sample (42nd month). Hypervariable genomic regions of HIV (V3 loop of the gp120) and HCV (HVR-1 on the E2 glycoprotein gene) were studied. In order to analyze genomic regions involved in different biological functions and with the cellular immune response, HCV core and NS5A were also chosen to be sequenced. Amplification of the different regions was done by RT-PCR and directly sequenced. Confirmation of sequences was done on reamplified material. Nucleotide sequences of the different time points were aligned with CLUSTAL W 1.5, and the corresponding amino acid ones were deduced.RESULTS: Hypervariable genomic regions of both viruses (HVR1 and gp120 V3 loop) presented several nonsynonymous changes but, while in the gp120 V3 loop mutations were detected in the sample obtained right after HCV superinfection and maintained throughout, they occurred following a sequential and cumulative pattern in the HVR1. In the NS5A region of HCV, two amino acid changes were detected during the follow-up period, whereas the core region presented several amino acid replacements, once the HCV chronic infection had been established.CONCLUSION: During the HIV-HCV superinfection, each genomic region analyzed shows a different evolutionary pattem.Most of the nucleotide substitutions observed are nonsynonymous and clustered in previously described epitopes,thus suggesting an immune-driven evolutionary process.

非洲猪瘟病毒入胞过程相关蛋白及分子机制的研究进展

非洲猪瘟病毒(ASFV)是非洲猪瘟的病原,可引发家猪和野猪急性、出血性死亡,目前尚无有效的疫苗和抗病毒药物。ASFV是囊膜病毒,其囊膜蛋白的结构和功能可能是影响病毒入侵和细胞嗜性的重要因素。病毒入胞是病毒感染细胞的第一步,通常是通过细胞表面特定的分子与病毒蛋白相结合吸附于宿主细胞表面,以ASFV入胞过程的病毒蛋白或宿主因子为靶点或可有效抑制ASFV的复制。从ASFV入胞过程中起重要作用的囊膜蛋白出发,了解ASFV的入胞分子机制,为ASFV入胞深入研究以及治疗性药物和疫苗的研发提供参考。

Baculovirus Surface Display of Zika Virus Envelope Protein Protects against Virus Challenge in Mouse Model

Zika virus(ZIKV)is emerging as a significant pathogen worldwide and may cause severe neurological disorders such as fetal microcephaly and Guillain-Barre syndrome.No drug or listed vaccines are currently available for preventing ZIKV infection.As a major target of neutralizing,ZIKV envelop(E)protein usually used for vaccine development.Nevertheless,the immunogenicity of ZIKV envelop(E)protein expressed by baculovirus display system has never been assessed.In this study,we reported a new strategy for surface display of ZIKV E protein by a recombinant baculovirus vector derived from Autographa californica multiple nuclear polyhedrosis virus(AcMNPV)and assessed its immunogenicity in mice.We produced recombinant fusion ZIKV E protein linked with signal peptide(SP)and transmembrane domain(TM)of AcMNPV GP64.The results showed that the recombinant protein was easy to produce by baculovirus display system.BALB/c mice immunized with this recombinant E protein developed ZIKV specific serum antibodies.The anti-E protein sera from the mice were able to effectively neutralize ZIKV in vitro.More importantly,AG6(IFN-a/b and IFN-c receptor deficient)mice immunized with recombinant E protein were protected against lethal ZIKV challenge.Together,thesefindings demonstrated that the recombinant E protein displayed by baculovirus can be conveniently prepared and displayed good immunogenicity in immunized mice.It is a promising practical approach for prompting the development of vaccine and related immunology research.

乙型肝炎病毒外膜大蛋白与HBV DNA的关系研究

目的:探讨乙型肝炎患者血清中乙型肝炎病毒外膜大蛋白(HBV-LP)与HBV DNA的关系。方法:采用酶联免疫吸附试验(ELISA)和实时荧光定量PCR法,检测320例乙型肝炎患者血清中的HBV-LP水平及HBV DNA载量。结果:HBV-LP水平与HBV DNA拷贝数变化相一致,两者呈正相关(r=0.949);不同模式的HBeAg血清中HBV-LP与HBV DNA阳性率,差异均无统计学意义(P>0.05)。结论:血清HBV-LP水平能反映乙型肝炎患者体内HBV复制程度,是判断HBV复制的血清学指标。

犬瘟热病毒分子生物学研究进展

犬瘟热病是由犬瘟热病毒 (CDV)感染犬和其他食肉动物的多发性、致死性传染病。CDV属于副黏病毒科麻疹病毒属有囊膜的负链线性RNA病毒 ,基因组长约 16kb。基因组中包括 6个非重叠基因区。它们的编码基因按 3′～ 5′端顺序依次为N P M F H L系列。最新研究表明CDV两个糖蛋白 (H ,F)是宿主免疫系统的主要目的抗原 ,产生中和抗体的重要抗原之一。N蛋白是保守性较强的免疫原性蛋白 ,此外N蛋白上含有T细胞表位。H 。

肾综合征出血热病毒50K结构蛋白免疫学特性的初步研究

以McAb亲和层祈纯化的HFRS病毒50K结构蛋白免疫BALB/c小鼠,证明其具有较好的免疫原性,可刺激产生中和抗体。该抗体不仅可中和细胞培养中的病毒,对HFRS病毒感染裸鼠也有一定保护作用。

乙型肝炎病毒外膜大蛋白检测与HBV—DNA及血清标志物的相关性分析

目的探讨乙型肝炎病毒外膜大蛋白（LHBs）与乙肝病毒血清标志物及HBV—DNA之间的相关性及临床意义。方法采用酶联免疫吸咐试验（ELISA）和实时荧光定量PCR法分别检测396例慢性乙型肝炎患者血清中LHBs，HBv-M和HBV—DNA并进行相关性分析。结果在HBeAg阳性的143例患者中HBV—DNA和LHBs的检出率分别为93．0％和96．5％；抗HBe阳性的191倒中，分别为63．9％和59．2％；在HBsAg阳性、抗HBc阳性的62例中分别为50．9％和38．6％；慢性乙型肝炎轻度193例，中度119例，重度84例，其乙肝病毒包膜大蛋白阳性率分别为57．2％，63．9％和59．5％，三组之间比较差异无统计学显著性意义（P〉0．05）。结论血清LHBs水平能够反映HBV感染者体内HBV—DNA复制程度，其灵敏度高于HBV—DNA，可作为病毒复制观察的新的血清学指标。

霍奇金淋巴瘤潜伏膜蛋白1、p53及bcl-2蛋白的表达及意义

目的探讨EB病毒潜伏膜蛋白(LMP1)、p53、bcl-2的蛋白表达在霍奇金淋巴瘤(HL)致病机制中的作用及其意义。方法对收集的31例石蜡包埋的HL组织进行LMP1、p53、bcl-2蛋白免疫组化染色。结果LMP1、p53、bcl-2的阳性率分别为58.1%、61.3%、35.5%。LMP1与p53相关性分析具有统计学意义。结论p53可能参与LMP1在HL致病机制中的作用,bcl-2可能与HL的致病关系不大。

丙型肝炎病毒E2蛋白作为酵母双杂交体系“饵”载体的构建及表达

目的 :用丙型肝炎 (丙肝 )病毒 (hepatitisCvirus,HCV)胞膜蛋白 2 (envelopeprotein 2 ,E2 )胞外区片段 ,构建酵母双杂交体系中“饵”载体 ,明确其在酵母细胞中的表达 ,排除自身激活作用 ,验证其能否用于酵母双杂交体系筛选cDNA文库。方法 :设计引物 ,从本室构建的含中国株Ⅲ型HCVE2片段的载体上 ,扩增出不包含C末端跨膜区的E2片段E2 6 6 1,经EcoRI和PstI双酶切后 ,克隆到酵母双杂交的“饵”载体pAS2 1质粒上 ,构建成载体pAS2 1 E2 6 6 1。阳性克隆经EcoRI和PstI双酶切鉴定后 ,转入酵母中 ,用酵母蛋白抽提物作Western杂交 ,证实其表达 ;滤膜印记杂交验证其有无自身激活作用。结果 :所构建的载体 pAS2 1 E2 6 6 1双酶切后 ,片段大小正确 ;转入酵母后 ,提取酵母质粒 ,PCR扩增出预期大小的片段 ,Western杂交出现阳性条带 ;滤膜杂交 ,转有 pAS2 1 E2 6 6 1和阴性对照pLAM5 ' 1的酵母菌落没有激活报告基因 ,而GAL4全长的阳性对照菌落变为蓝色。结论 :含有HCVE2 6 6 1的“饵”载体构建正确 ,在酵母细胞中能表达出E2蛋白 ,并且没有自身激活报告基因的功能 ,能够应用于酵母双杂交体系。

丙型肝炎病毒包膜蛋白E2抗原模拟表位的筛选和鉴定

目的 :筛选丙肝病毒 (HCV)包膜蛋白 (E2 )特异性噬菌体模拟表位。方法 :以抗 HCVE2的单克隆抗体作为固相筛选分子 ,对噬菌体随机七肽库进行 5轮“吸附 洗脱 扩增”的筛选过程 ,随机挑取 5 0个克隆 ,经酶联免疫吸附法 (ELISA)鉴定、交叉反应以及竞争抑制性实验 ,最后对所选克隆进行DNA序列分析 ,以确定HCVE2抗原的模拟表位。结果 :经噬菌体富集后 ,得到 12个阳性克隆 ,确定氨基酸序列XXPXYXW为HCVE2的模拟表位。结论 :用噬菌体七肽库成功筛选得到HCVE2的模拟表位 ,为开展用HCV模拟表位探索HCV的防治研究创造了条件。

IFN-γ与HIV-1gp120融合蛋白的表达及其免疫调节作用

目的:研究gamm a干扰素(IFN-γ)对人类免疫缺陷病毒(H IV-1)gp120蛋白免疫小鼠后效果的调节作用。方法:利用分子生物学技术,构建表达中国流行株H IV-1gp 120 N端片段基因的原核质粒p et44b-gp 120,及共表达H IV-1gp 120 N端片段基因与IFN-γ基因的原核质粒p et44b-gp 120/IFN-γ,在E.coli BL 21(DE 3)细胞中进行低温诱导蛋白表达,然后经纯化后免疫BALB/c小鼠。实验设皮下注射gp120/IFN-γ组、gp120组和PBS三组,以检测H IV-1gp120诱导的T细胞增殖能力,CTL杀伤作用以及Th1型细胞因子(IL-2,IFN-γ)和Th2型细胞因子(IL-4,IL-10)的表达情况。结果:通过PAGE和W estern b lot检测,证实上述两种蛋白在DE 3细胞中得到表达。免疫学实验表明,针对H IV-1 gp120的特异性T淋巴细胞增殖作用、特异性CTL杀伤作用,以及Th1型细胞因子IL-2和IFN-γ表达在三组之间均有显著性差异(P<0.05),gp120/IFN-γ组高于gp120组,gp120组高于PBS组(P<0.05)。反映体液免疫反应的Th2型细胞因子IL-4和IL-10的表达在三组之间没有显著性差异(P>0.05)。结论:协同应用IFN-γ基因可明显加强H IV-1gp 120基因的细胞免疫反应。

HBV包膜蛋白N-糖基化修饰变异与HBV相关慢加急性肝衰竭发生和转归的关系

目的探索HBV包膜蛋白N-糖基化修饰变异与慢性乙型肝炎患者发生慢加急性肝衰竭(ACLF)的关系。方法入选HBV-ACLF患者90例,同期60例慢性乙型肝炎作对照。套式PCR扩增患者的HBV S区序列并测序,分析HBV包膜蛋白N-糖基化情况与ACLF发生的关系,并分析ACLF疾病严重程度、90 d生存率与HBV包膜蛋白N-糖基化的关系。结果慢性乙型肝炎患者纳入分析51例(85.0%),HBV-ACLF患者纳入分析79例(87.8%)。慢性乙型肝炎患者Asn59 N-糖基化样本33例(64.7%),Asn4 N-糖基化样本21例(41.2%),高于HBV-ACLF患者的Asn59 N-糖基化样本16例(20.3%)(P<0.001)和Asn4 N-糖基化样本14例(17.7%)(P=0.003)。ACLF患者中Asn59、Asn4 N-糖基化与非糖基化患者肝功能水平、HBV DNA、MELD评分及90 d生存率差异均无统计学意义(P值均>0.05)。结论 HBV包膜蛋白变异导致的Asn59、Asn4 N-糖基化水平降低与ACLF发生可能相关。

HCV E2和HBV preS融合蛋白真核表达载体的构建及在哺乳动物细胞中的表达

目的：构建丙型肝炎病毒（ＨＣＶ）Ｅ２和乙型肝炎病毒（ＨＢＶ）ｐｒｅＳ融合蛋白的真核表达载体。方法：用ＰＣＲ方法扩增ＨＢＶｐｒｅＳ和ＨＣＶＥ２蛋白基因，并将二者克隆到真核表达载体ｐｃＤＮＡ３中，用脂质体转染试剂将其转入ＣＯＳ７细胞，通过免疫荧光检测细胞瞬时表达的融合蛋白。结果：Ｅ２ｐｒｅＳ融合蛋白基因包括了全长的ＨＢＶｐｒｅＳ和ＨＣＶＥ２蛋白基因，嵌合基因长度约１．６ｋｂ。表达载体在ＣＯＳ７细胞表达出了Ｅ２ｐｒｅＳ融合蛋白。结论：构建的Ｅ２ｐｒｅＳ融合蛋白表达载体能在哺乳动物细胞中表达融合蛋白。

牛病毒性腹泻病毒囊膜蛋白E2基因不同高可变区缺失型核酸疫苗的构建

为了构建牛病毒性腹泻病毒(Bovine viral diarrhea virus,BVDV)囊膜蛋白E2基因高可变区(hyper variable region,HVR)缺失型核酸疫苗,为BVDV防控提供新的方法,试验依据GenBank中BVDV基因组序列设计引物,以BVDV新疆分离株TC cDNA为模板,扩增E2基因并测序,使用生物信息学软件分析E2蛋白的跨膜区和HVR区域等;采用常规PCR和搭桥PCR等方法扩增E2基因的不同缺失型HVR区域,并将其插入到真核表达载体pVAX1中,构建成真核表达质粒;分别于0,2,4周时免疫Balb/c小鼠,并在首次免疫后0,14,28,42天采集血清,通过ELISA法测定IgG水平。结果表明:成功扩增出BVDV TC株E2基因,大小为1122 bp;E2基因与BVDV 1b毒株HP-KY-PK13(登录号KT355592.1)及BVDV 1b毒株3156(登录号JN704144.1)的E2基因具有较高的同源性,E2 HVR1区域位于E2基因N端,HVR2区域位于E2基因中间部位;成功构建了E2基因不同的HVR缺失型真核表达质粒pVAX1-E2 HVR1△1-20、△1-64、△21-40、△41-64和pVAX1-E2 HVR2△140-216;与免疫pVAX1相比,E2基因不同HVR缺失质粒及野生型E2质粒诱导的IgG抗体水平显著或极显著升高(P<0.05或P<0.01);而在首次免疫42天时,用pVAX1-E2 HVR1△41-64免疫诱导的IgG抗体水平最高,约为pVAX1-E2的3.9倍。说明用pVAX1-E2 HVR1△41-64制备的核酸疫苗能诱导动物产生较高水平的IgG。

HCV包膜蛋白E2的表达及其抗原性的检测

为研究丙型肝炎病毒 (HCV)包膜蛋白E2在原核细胞和真核细胞中的表达 ,并对表达蛋白的抗原性进行检测。将HCVE2区N端的一段基因分别克隆入原核表达载体pQE30和真核表达载体pEF1/HisC中进行E2蛋白的表达 ,采用ELISA和WB对表达蛋白的抗原性进行检测。结果在原核细胞和真核细胞中均表达了预期大小的蛋白 ,能同HCV感染者血清发生特异性反应。提示该段E2基因在原核表达系统和真核表达系统均能表达出具有抗原活性的蛋白 ,其中真核细胞表达的蛋白可被糖基化。

丙型肝炎病毒E2蛋白的高效表达及其临床应用

目的：在大肠杆菌中表达丙型肝炎病毒（ｈｅｐａｔｉｔｉｓ Ｃｖｉｒｕｓ，ＨＣＶ）Ｅ２ 包膜蛋白，并对其进行初步纯化。用纯化的包膜蛋白检测ＨＣＶＲＮＡ 阳性血清中Ｅ２ 抗体，研究其检测Ｅ２ 抗体的敏感性和特异性。方法：将ＨＣＶＥ２ 蛋白基因克隆到原核表达载体中，并转化大肠杆菌。用ＩＰＴＧ 诱导细菌表达Ｅ２ 蛋白，经离子交换层析纯化蛋白。用纯化的包膜蛋白包被微孔板，经ＥＬＩＳＡ方法检测ＨＣＶＲＮＡ阳性血清中的Ｅ２ 抗体。结果：在大肠杆菌表达了相对分子质量为４５ ０００ 的可溶性Ｅ２ 蛋白，占细菌总蛋白量的２１％ ，纯化后，蛋白纯度为８５％ 。用其检测ＨＣＶ 患者血清１７１ 例，Ｅ２ 抗体的检出率为６３％ 。在Ｅ２ 抗体阳性的血清中，部分为抗ＨＣＶ 阴性。结论：Ｅ２ 蛋白的表达及初步纯化的成功，可能用于临床检测。各基因型ＨＣＶＥ２ 包膜蛋白的抗体之间有交叉反应，在目前的抗ＨＣＶ 诊断试剂中加入可溶性、非融合Ｅ２ 包膜蛋白，可以改善抗ＨＣＶ 诊断试剂的特异性和敏感性。

丙型肝炎病毒E2基因激发小鼠细胞免疫的实验研究

目的 :研究丙型肝炎病毒E2基因接种能否诱导机体产生细胞免疫以及乙型肝炎病毒preS基因对其诱导细胞免疫能力的影响。方法 :以BALB/c小鼠的骨髓瘤细胞株 (SP2 /O)为宿主细胞 ,建立表达丙肝病毒E2蛋白的靶细胞 ,将其注射于E2基因或 preS与E2融合蛋白基因免疫的小鼠腹腔 ,记录小鼠接种瘤细胞后的存活期 ,以小鼠存活期的长短作为反映基因免疫小鼠对HCVE2蛋白细胞免疫的有无或强弱的指标。结果 :靶细胞表达了相对分子质量约为 70 0 0 0的E2蛋白 ,E2基因接种小鼠的存活期长于对照组 ,单独E2基因接种组的存活期显著长于对照组 ,也长于 preS与E2融合蛋白基因免疫组。 结论 :E2基因接种能诱导细胞免疫 ,但与 preS融合后 ,其诱导细胞免疫的能力会降低。