乙型肝炎病毒核心抗原相关抗原(HBcrAg)的研究进展

HBcrAg包括HBcAg、HBeAg、p22cr蛋白,在HBV感染中具有重要的临床意义.本文旨在对近年来HBcrAg的概念、检测方法及其在乙型肝炎诊治中的检测意义等方面的进展作一综述.     

抗⁃HBc定量测定在乙型肝炎病毒慢性感染性疾病中的应用

乙肝病毒核心抗体(抗?HBc)与机体免疫相关,在现症及既往乙型肝炎病毒(HBV)感染者血清中均可测得,其在HBV自然史的判断及治疗策略中可发挥巨大作用.HBV感染慢性化与肝内共价闭合环状DNA(cccDNA)活跃转录和持续存在密切相关;抗?HBc定量与cccDNA水平有较好相关性,可作为综合评价HBV感染性疾病的一项指...     

应用单克隆抗HBc和抗人IgM(μ链特异)检测血清抗HBcIgM试剂盒的研究

本试剂盒以自制羊抗人IgM(μ链特异)为包被抗体,鼠单克隆抗HBcIgG_3的酶结合物为指示抗体,并采用HBsAg携带者非肝炎死者肝脏提取的HBcAg。经166份病人及健康人血清1:1,000稀释后,用丹麦Dako公司和美国Sigma公司抗人IgM(μ链特异产品)包被微板进行比较,以Dako公司抗人IgM检测结果为相对标准,初步证明:自制抗人IgM与Sigma公司产品不论是相对灵敏度,相对特异性,相对预报率还是相对符合率等四个指标,X~2测定结果并无显著差别。己初步用于临床实验室诊断,结果满意。     

用ELISA微板法检测乙型肝炎病毒核心抗原

以双抗体包被的抗体夹心法，用微板ＥＬＩＳＡ检测血清乙型肝炎病毒核心抗原（ＨＢＣＡｇ），确定双包被工作浓度ＭＣ－抗－ＨＢｃ（效价１０００）为０．０４μｌ／孔，ＭＣ－抗－ＨＢｓ（１ｍｇ／ｍｌ）为３～４μｌ／孔；最佳裂解剂及其工作浓度为７％ＮＰ－４０巯基乙醇溶液。分别用不同的酶标记抗体检测，均证明双包被具有特异性。加入抗－ＨＢｃ进行阻断试验，其阻断率为７９．３％。对８４４例ＨＢｓＡｇ阴性的血清及１１４例ＨＢＶ－ＤＮＡ探针阴性血清用本法进行ＨＢｃＡｇ检测，均为阴性。在临床应用上，本法的阳性率明显高于试管法的，与ＨＢＶ－ＤＮＡ探针的阳性符合率为９１．４％，并且特异性与ＨＢＶ－ＤＮＡ探针的一致。     

在枯草杆菌中产生乙型肝炎病毒核心抗原和口蹄疫病毒的主要抗原

用枯草杆菌来制造病毒或真核生物的多肽,有其优越性:它是非病原菌;不象大肠杆菌那样产生内毒素与大量分泌胞外蛋白,此外它已被广泛用于酶与抗菌素,因此现有的大规模培养枯草杆菌,并从其培养物中分离蛋白质的技术可资利用。为了在枯草杆菌中表达编码口蹄疫病毒的主要抗原(VPI)基因,应用了两个质粒:含有抗红霉素基因的pBD9以及含有编码全部VPI(除其NH_2     

丙型肝炎病毒核心抗原在血液透析患者中的检测和应用

Objective To investigate the utility of the HCV core antigen ELISA in the detection of HCV infection in hemodialysis patients and to compare with Anti-HCV antibodies 3rd generation ELISA.Methods Two hundred fifty hemodialysis patients were included in the study. Anti-HCV antibodies and total HCVcAg was determined by ELISA kits. HCV RNA was determined using reverse transeriptase polymerase chain reaction (RT-PCR) .Results Forty-three out of 250 (17.2%) patients were positive for anti-HCV antibodies and 18% WERE positive for HCVcAg. 13/250 (5.2%) were positive for HCVcAg but anti-HCV negative, All 13 were confirmed viremic by in-house nested RT-PCR leading to specificity of 100%. Viral load of (49258±28682) copies/ml were detected in HCVcAg positive cases was higher in comparison to (23938±10780) copies/ml in the anti-HCV positive group (P < 0.05). The viral load of 4 negative cases for HCVcAg assay but anti-HCV positive was 306±161 copies/rnl which was significantly lower in comparison to HCVcAg positive group (P < 0.001). Conclusion Detection of total HCVcAg together with anti-HCV will be useful for patients undergoing hemodialysis who have a longer window period due to immunosuppressed state. HCVcAg was compatible for the HCV RNA in serum and total HCVcAg ELISA is beth a cost-effective and a less labor-intensive alternative to PCR, enhancing its clinical utility.     

丙型肝炎病毒核心抗原在血液透析患者中的检测和应用

Objective To investigate the utility of the HCV core antigen ELISA in the detection of HCV infection in hemodialysis patients and to compare with Anti-HCV antibodies 3rd generation ELISA.Methods Two hundred fifty hemodialysis patients were included in the study. Anti-HCV antibodies and total HCVcAg was determined by ELISA kits. HCV RNA was determined using reverse transeriptase polymerase chain reaction (RT-PCR) .Results Forty-three out of 250 (17.2%) patients were positive for anti-HCV antibodies and 18% WERE positive for HCVcAg. 13/250 (5.2%) were positive for HCVcAg but anti-HCV negative, All 13 were confirmed viremic by in-house nested RT-PCR leading to specificity of 100%. Viral load of (49258±28682) copies/ml were detected in HCVcAg positive cases was higher in comparison to (23938±10780) copies/ml in the anti-HCV positive group (P < 0.05). The viral load of 4 negative cases for HCVcAg assay but anti-HCV positive was 306±161 copies/rnl which was significantly lower in comparison to HCVcAg positive group (P < 0.001). Conclusion Detection of total HCVcAg together with anti-HCV will be useful for patients undergoing hemodialysis who have a longer window period due to immunosuppressed state. HCVcAg was compatible for the HCV RNA in serum and total HCVcAg ELISA is beth a cost-effective and a less labor-intensive alternative to PCR, enhancing its clinical utility.     

B型肝炎表面抗原和核心抗原在大肠杆菌中的合成

近来人们发现22—nm颗粒型B型肝炎表面抗原(HBsAg)在临床上具有一定的疗效。但是,这种颗粒型的HBsAg是从慢性肝炎患者血清中制备的,数量甚微。重组DNA法为大量生产该种疫苗开辟了广     

丙型肝炎病毒核心抗原在血液透析患者中的检测和应用

Objective To investigate the utility of the HCV core antigen ELISA in the detection of HCV infection in hemodialysis patients and to compare with Anti-HCV antibodies 3rd generation ELISA.Methods Two hundred fifty hemodialysis patients were included in the study. Anti-HCV antibodies and total HCVcAg was determined by ELISA kits. HCV RNA was determined using reverse transeriptase polymerase chain reaction (RT-PCR) .Results Forty-three out of 250 (17.2%) patients were positive for anti-HCV antibodies and 18% WERE positive for HCVcAg. 13/250 (5.2%) were positive for HCVcAg but anti-HCV negative, All 13 were confirmed viremic by in-house nested RT-PCR leading to specificity of 100%. Viral load of (49258±28682) copies/ml were detected in HCVcAg positive cases was higher in comparison to (23938±10780) copies/ml in the anti-HCV positive group (P < 0.05). The viral load of 4 negative cases for HCVcAg assay but anti-HCV positive was 306±161 copies/rnl which was significantly lower in comparison to HCVcAg positive group (P < 0.001). Conclusion Detection of total HCVcAg together with anti-HCV will be useful for patients undergoing hemodialysis who have a longer window period due to immunosuppressed state. HCVcAg was compatible for the HCV RNA in serum and total HCVcAg ELISA is beth a cost-effective and a less labor-intensive alternative to PCR, enhancing its clinical utility.     

丙型肝炎病毒核心抗原在血液透析患者中的检测和应用

Objective To investigate the utility of the HCV core antigen ELISA in the detection of HCV infection in hemodialysis patients and to compare with Anti-HCV antibodies 3rd generation ELISA.Methods Two hundred fifty hemodialysis patients were included in the study. Anti-HCV antibodies and total HCVcAg was determined by ELISA kits. HCV RNA was determined using reverse transeriptase polymerase chain reaction (RT-PCR) .Results Forty-three out of 250 (17.2%) patients were positive for anti-HCV antibodies and 18% WERE positive for HCVcAg. 13/250 (5.2%) were positive for HCVcAg but anti-HCV negative, All 13 were confirmed viremic by in-house nested RT-PCR leading to specificity of 100%. Viral load of (49258±28682) copies/ml were detected in HCVcAg positive cases was higher in comparison to (23938±10780) copies/ml in the anti-HCV positive group (P < 0.05). The viral load of 4 negative cases for HCVcAg assay but anti-HCV positive was 306±161 copies/rnl which was significantly lower in comparison to HCVcAg positive group (P < 0.001). Conclusion Detection of total HCVcAg together with anti-HCV will be useful for patients undergoing hemodialysis who have a longer window period due to immunosuppressed state. HCVcAg was compatible for the HCV RNA in serum and total HCVcAg ELISA is beth a cost-effective and a less labor-intensive alternative to PCR, enhancing its clinical utility.     

大肠杆菌LPS和麻疹病毒特异性抗体定量测定法的建立和在免疫缺陷病患者中的初步应用

采用ＬｅｉｖｅＥＤＴＡ法及Ｃｅｆａｖｌｏｎ法制备的特异大肠杆菌ＬＰＳ，麻疹病毒抗原建立了特异抗体ＥＬＩＳＡ定量法。此法灵敏度高，特异性强，实验方法稳定。对于免疫缺陷病人的观察治疗具有指导意义。     

小鼠造血干细胞因子的cDNA克隆化及在大肠杆菌表达

造血干细胞因子（ＳＣＦ）是一种多功能造血生长因子，与其它造血生长因子协同作用，刺激不同分化阶段的造血细胞增殖和分化。本文采用ＲＴ－ＰＣＲ技术从新生ＢＡＬＢ／Ｃ小鼠胸腺组织克隆了ＳＣＦ膜外功能区ｃＤＮＡ，进而在大肠杆菌表达出具有生物学活性的重组蛋白。     

抗隐孢子虫子孢子ScFv-PE40免疫毒素表达质粒的构建及其在大肠杆菌中的表达

用PCR方法扩增抗隐孢子虫子孢子ScFv片段,插入表达载体pET-28a中,构建重组质粒pET-28-ScFv,然后,将绿脓杆菌外毒素(PE40)片段定向克隆到ScFv基因的下游,构建免疫毒素表达质粒pET28ScFv-PE40。经酶切分析、PCR检测和测序进行鉴定。成功地构建了免疫毒素表达质粒pET28ScFv-PE40。将上述质粒转化受体菌BL21(DE3)后,经IPTG诱导,成功地表达了目的蛋白。免疫毒素ScFv-PE40大小约为66kDa,表达量约占菌体蛋白总量的14%。     

人孕激素受体在大肠杆菌中的高效表达及应用

为了制备抗重组人孕激素受体单克隆抗体，用聚酶链反应扩增人孕激素受体氨基础编码区段，定位克隆连入主同效表达载体ＰＭＳ－３１ｂ，构建重组质粒ＰＭＳ－ＰＲａ，将其转入大肠杆菌ＰＯＰ＾２１３６，     

免疫沉淀法检测RNPs的RNA组分及其应用

本文用免疫沉淀法检测各种RNPs的RNA组分,发现各种抗RNPs抗体所沉淀的RNAs是具有特征性的,抗U_1RNP抗体沉淀出U_1RNA,抗Sm抗体沉淀出U_2RNA、U_1RNA、U_4RNA、U_5RNA和U_6RNA,抗SSA抗体沉淀出了Y_1-Y_5RNAs,抗SSB抗体沉淀出Y_1-Y_5RNAs、La4.5RNA和7-2RNA,抗JO-1抗体沉淀出tRNA~(His)。因而根据待测血清所沉淀的RNAs可判断血清中各种抗BNPs抗体,结果显示,免疫沉淀法较免疫双扩散法敏感。     

白细胞介素8片段在大肠杆菌中表达及其功能研究

利用ＤＮＡ重组技术在大肠杆菌中表达出白细胞介素８的Ｎ端４３氨基酸片段（ＩＬ８－Ｎ）和Ｃ端２４氨基酸片段（ＩＬ８－Ｃ），经过ＦＡＣＳ和趋化活性鉴定表明，ＩＬ８－Ｎ和ＩＬ８－Ｃ均能与中性粒细胞结合，其中ＩＬ８－Ｎ能部分封闭ＩＬ－８的趋化活性。     

弓形虫ROP2基因的克隆及在大肠杆菌中的表达

目的 :构建弓形虫棒状体蛋白 (ROP2 )基因重组质粒并在大肠杆菌中表达 ,用于筛选弓形虫新的诊断抗原和疫苗分子。方法 :根据ROP2基因已知序列 ,设计合成一对引物 ,用PCR方法从弓形虫RH株基因组DNA中扩增出ROP2基因片段 ,再克隆到pGEX 4T 1质粒 ,重组质粒经酶切及PCR鉴定 ,而后进行测序 ;重组质粒在大肠杆菌BL2 1 (DE3)中进行ITPG诱导表达 ,表达产物经SDS PAGE及WesternBlot分析。结果 :ROP2基因PCR产物大小与预期相符 ,约 1 0 4 3bp ,重组质粒经酶切及PCR鉴定表明获得正确重组子 ,测序结果与已知序列基本吻合 ;SDS PAGE及免疫印迹显示ROP2融合蛋白表观分子量约为 5 5kDa ,表达产物约占菌体总蛋白1 7% ,具有一定的免疫反应性。结论 :克隆并表达了弓形虫ROP2基因 ,为下一步弓形虫诊断及疫苗研究奠定了基础     

乙型肝炎病毒preS抗原在大肠杆菌中的高效表达与鉴定

利用ＰＣＲ和基因重组技术构建了乙型肝炎病毒完整ｐｒｅＳ抗原高效表达克隆，该克隆在大肠杆菌中表达一分子量约３１ｋＤ的融合蛋白，由ＭＳ２、白细胞介素３Ｎ端１４个氨基酸接头和ｐｒｅＳ抗原组成，在ＩＬ－３Ｎ端与ＭＳ２连接处有凝血酶识别位点，可被该酶切开。     

登革2型病毒43株膜蛋白前体基因片段的克隆与表达

依照国际标准株ＮＧＣ的序列，设计合成了一对引物，用于扩增登革２型病毒中国分离株Ｄ２－４３的ＰｒＭＣ端抗原区的基因片段，结构分析及特异性分析的结果表明引物合乎要求，反转录（ＲＴ）－ＰＣＲ扩增出一３８５ｂｐ的目的片段，经ＨａｅⅢ酶切得到２１９、１６６ｂｐ的两条预期片段，表明ＲＴ－ＰＣＲ扩增出ＰｒＭ基因片断。扩增产物经ＢａｍＨＩ酶切后插入经ＳｍａＩ、ＢａｍＨＩ双酶切的ｐＵＥｘ１中，转化ＭＣ１０６１菌，表达与β－半乳糖苷酶的融合蛋白，ＳＤＳ－ＰＡＧＥ结果表明有一约１３０ｋＤ的目的蛋白带，表达量占菌体总蛋白的３０％，Ｗｅｓｔｅｒｎｂｌｏｔ及ＥＬＩＳＡ结果表明表达产物能与Ｄｅｎｇｕｅ－２多抗血清结合，为ＰｒＭ的免疫学及生物学性质研究打下了基础。