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 diagnositic 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 El(Gl). one from E2 (G31), three from NS3 (G6, G61, G7),one from NS5B (G821) and one chimeric fragment from NS3and NS5B (G61 821) could be expressed well and showed obvious immunoreactivity by Western blotting.One clone with I-KGV 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 Gl, G,31, G61, G821 and G61 821were detected in indirected ELISA as coating antigen respectively. Only recombinant Gl 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 El, E2. NS3 and NS5 fragment of HGV contain antigenic epitopes, which could be produced in prokaryotically expressed recombinant proteins. A high. yield recombinant protein (Gl) 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.

C反应蛋白联合血常规检测对肺炎疾病的诊断价值

目的:探讨C反应蛋白(CRP)联合血常规检测对肺炎疾病的诊断价值。方法:回顾性分析2022年1—12月普宁市人民医院收治的100例肺炎患者的临床资料,根据感染类型分为细菌性肺炎组和病毒性肺炎组,各50例。患者均进行血常规检测、CRP检测。比较两组血常规指标[白细胞计数(WBC)、中性粒细胞计数(N)、中性粒细胞/淋巴细胞比值(NLR)、血小板计数(PLT)、平均血小板体积(MPV)、淋巴细胞计数(L)]、CRP水平,分析CRP联合血常规检测对细菌性肺炎的诊断效能。结果:细菌性肺炎组WBC、N及NLR水平高于病毒性肺炎组,差异有统计学意义(P<0.001);细菌性肺炎组CRP水平高于病毒性肺炎组,差异有统计学意义(P<0.001)。CRP联合血常规检测的AUC为0.973,高于各项单一检测,差异有统计学意义(P<0.05)。结论:CRP联合血常规检测对细菌性肺炎有一定的诊断价值,有助于细菌性肺炎和病毒性肺炎的早期鉴别诊断。

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

AIM: To study HCV polyprotein processing is important forthe understanding of the natural history of HCV and thedesign of vaccines against HCV. The purpose of this studyis to investigate the affection of context sequences onhepatitis C virus (HCV) E2 processingMETHODS: HCV genes of different lengths were expressedand compared in vaccinia virus/T7 system with homologouspatient serum S94 and mouse anti-serum ME2116 raisedagainst 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( ～ 75kDa end ～ 60kDa) were detected using S94 and ME2116,respectively. Deglycosylation analysis showed that thisdifference was mainly due to different glycosylation. Endo Hresistance and its failure to bind to GNA lectin demonstratedthat the higher molecular weight form (75kDa) of E2 wascomplex-type glycosylated, which was readily recognized byhomologous patient serum S94. Expression of complex-typeglycosylated E2 could not be detected in all of the core-truncated constructs tested, but readily detected inconstructs encoding full-length core sequences.CONCLUSION: The upstream conserved full-length corecoding sequence was required for the production of E2glycoproteins carrying complex-type N-glycans whichreacted strongly with homologous patient serum andtherefore possibly represented more mature forms of E2. Ascomplex-type N-glycans indicated modification by Golgienzymes, the results suggest that the presence of full-lengthcore might be critical for E1/E2 complex to leave ER. Ourdata may contribute to a better understanding of theprocessing of HCV structural proteins as well as HCVmorphogenesis.

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.

hs-CRP、IL-6和WBC检测对小儿呼吸道病毒感染的诊断价值

目的 探讨超敏C-反应蛋白(hs-CRP)、白细胞介素-6(IL-6)、白细胞(WBC)计数检测对小儿呼吸道病毒感染的诊断价值。方法 选取2020年9月至2022年2月收治的50例小儿呼吸道感染患儿为研究对象(病毒感染组),另选50例同期健康儿童为对照组。比较2组hs-CRP、IL-6、WBC水平及一般资料,分析小儿呼吸道病毒感染危险因素,并绘制ROC曲线。结果 病毒感染组患儿hs-CRP、IL-6、D-二聚体、降钙素原(PCT)、WBC、血清淀粉样蛋白(SAA)水平显著高于对照组(P<0.05);病毒感染组居住农村、有早产史、哮喘史患儿占比显著高于对照组(P<0.05);小儿呼吸道病毒感染时hs-CRP、IL-6、WBC水平升高,且居住农村、有早产史及哮喘史是小儿呼吸道病毒感染的危险因素(P<0.05);hs-CRP、IL-6、WBC及联合诊断小儿呼吸道病毒感染的曲线下面积AUC分别为0.644、0.885、0.617、0.923,联合诊断AUC显著高于单独检测(P<0.05)。结论 hs-CRP、IL-6、WBC在小儿呼吸道病毒感染患儿中水平较高,联合检测诊断价值较高。

白细胞计数、中性粒细胞与淋巴细胞比值联合红细胞沉降率、C反应蛋白在病毒性肺炎与细菌性肺炎诊断鉴别中的应用价值

目的研究白细胞计数(WBC)、中性粒细胞与淋巴细胞比值(NLR)联合红细胞沉降率(ESR)、C反应蛋白(CRP)在病毒性肺炎与细菌性肺炎诊断鉴别中的应用价值。方法选取2019年2月至2021年12月安徽省第二人民医院54例病毒性肺炎(病毒组)、54例细菌性肺炎(细菌组)作为研究对象。比较两组血小板(PLT)、平均血小板体积(MVP)、WBC、N、L、NLR、ESR、CRP水平;分析WBC、NLR联合ESR、CRP检测对细菌性肺炎的诊断效能。结果细菌组WBC、N、NLR高于病毒组(P<0.05);两组PLT、MPV、L比较,差异无统计学意义(P>0.05)。细菌组ESR、CRP水平高于病毒组(P<0.05)。联合检测的曲线下面积高于各项单一检测(P<0.05)。结论WBC、NLR联合ESR、CRP检测能准确鉴别病毒性肺炎与细菌性肺炎,应用价值较高。

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。

降钙素原测定在婴幼儿呼吸道感染诊治中的临床意义

目的探讨血清降钙素原(PCT)、C反应蛋白(CRP)、白细胞(WBC)测定在儿童感染性疾病中的临床意义。方法将住院的84例患儿分成细菌感染、病毒感染和非感染对照三组,检测各组血清PCT、CRP、WBC的含量,并对各指标表达量和阳性率进行比较。结果细菌感染组和病毒感染组PCT分别为7.81±12.51,0.31±0.45,两组含量差异有统计学意义(P<0.05);WBC在两组表达量分别为15.24±6.06,10.88±4.68,两组比较差异有统计学意义(P<0.05),CRP在两组含量分别为28.03±31.91,15.46±19.35,两组比较差异无统计学意义(P>0.05)。PCT在细菌组、病毒组、非感染组阳性率比较差异有统计学意义(P<0.01);CRP在细菌组和病毒组阳性率比较差异无统计学意义(P>0.05),对照组和其他两组间比较差异有统计学意义(P<0.05);WBC在对照组和病毒组阳性率比较差异无统计学意义(P>0.05),细菌组和其他两组间比较差异有统计学意义(P<0.05)。结论血清PCT可作为儿童感染性疾病的鉴别诊断与早期诊断的有效指标。

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。

扶正化瘀胶囊对瘀血阻络肝肾不足型慢性乙型病毒性肝炎肝纤维化患者外周血单个核细胞中TGF-β_1/BMP-7比值的影响

目的观察慢性乙型病毒性肝炎肝纤维化瘀血阻络肝肾不足证型患者在拉米夫定抗病毒的基础上,加用扶正化瘀胶囊抗肝纤维化治疗前后肝功能、乙型肝炎病毒脱氧核糖核酸(hepatitis B virus deoxyribo-nucleicacid,HBVDNA)、肝组织病理学、外周血单个核细胞(peripheral blood mononuclear cell,PBMC)中转化生长因子β1/骨形成蛋白7(transforming growth factor-beta1/bone morphogenetic protein-7,TGF-β1/BMP-7)比值的变化,以及HBVDNA酪氨酸-甲硫氨酸-天门冬氨酸-天门冬氨酸(tyrosine-methionine-aspartate-aspar-tate,YMDD)变异的情况。方法选取瘀血阻络型慢性乙型病毒性肝炎肝纤维化患者80例,分为两组,对照组43例单用拉米夫定治疗,治疗组37例采用拉米夫定加扶正化瘀胶囊治疗,疗程6个月,疗程结束后均继续给予拉米夫定治疗,随访6个月。治疗前后进行肝组织病理检查,采用荧光定量PCR方法测定血清HBV DNA、TGF-β1和BMP-7。随访结束时检测HBV DNA YMDD变异。结果治疗6个月后两组在降低谷丙转氨酶(ALT)、谷草转氨酶(AST)、HBV DNA方面均有很好的疗效,与治疗前比较差异有统计学意义(P<0.05),但组间比较差异无统计学意义。随访6个月发现,对照组有9例出现YMDD变异,治疗组有5例出现YMDD变异,治疗组显著低于对照组(P<0.05)。TGF-β1/BMP-7比值治疗组显著低于对照组(0.09vs0.25,P<0.05)。在肝组织病理学改变方面,治疗组炎症活动度G3以上及纤维化程度S3以上所占比率显著低于对照组(P<0.05)。结论慢性乙型病毒性肝炎肝纤维化瘀血阻络肝肾不足型患者在拉米夫定抗病毒治疗的基础上,加用扶正化瘀胶囊可以降低HBV DNA变异,改善肝组织炎症及纤维化,并可能通过降低TGF-β1/BMP-7比值参与抗肝纤维化。

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。

CRP和WBC联合检测在儿童急性呼吸道感染中的应用价值

目的探讨C-反应蛋白(CRP)测定和白细胞(WBC)计数在儿童急性呼吸道感染中的应用价值。方法选取该院儿科住院的急性呼吸道感染患儿230例,其中细菌感染组130例,病毒感染组50例,肺炎支原体感染组50例,并以50例健康体检儿童作为对照组,分别检测外周血的CRP和WBC。结果细菌感染组的CRP阳性率明显高于WBC的阳性率。各感染组的CRP和WBC阳性率,细菌感染组与对照组相比差异有统计学意义(P<0.05),病毒感染组与对照组相比差异无统计学意义(P>0.05),肺炎支原体感染组与对照组相比,CRP阳性率差异无统计学意义(P>0.05),WBC阳性率差异有统计学意义(P<0.05)。结论 CRP和WBC联合检测对儿童急性呼吸道感染类型的鉴别有重要价值。

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。