Ⅰ型单纯疱疹病毒糖蛋白G基因片段的克隆表达及初步鉴定

通过计算机分析Ⅰ型单纯疱疹病毒糖蛋白G的氨基酸序列,筛选出HSV1gG蛋白中优势抗原决定簇位点,用PCR技术扩增克隆含强抗原决定簇较集中的基因片段。将该段基因克隆至质粒表达载体pGEX4T2内,转化大肠杆菌TG1,构建成功了高效表达Ⅰ型单纯疱疹病毒糖蛋白G基因的工程菌。用纯化的表达蛋白HSV1gGGST作抗原ELISA分析证实有较好的抗原性和特异性,显示可应用于单纯疱疹病毒感染的诊断 。     

SARS病毒S和N蛋白抗原表位的基因合成、融合表达及纯化鉴定

通过计算机分析SARS病毒N蛋白和S蛋白的氨基酸序列 ,初步确定含强抗原表位的N蛋白片段和S蛋白片段 ,共 5 6 0个氨基酸。选择真核和原核生物均偏爱的密码子 ,化学合成全新的SARS病毒N蛋白片段和S蛋白片段的基因序列 ,利用基因工程技术将两个基因片段串联 ,克隆至质粒Pet2 8a(+)内的NcoⅠ/EcoRⅠ位点 ,表达S蛋白片段和N蛋白片段的融合蛋白。将重组质粒转化大肠杆菌BL21(DE3) ,筛选获得了高效表达SARS病毒S蛋白片段和N蛋白片段融合蛋白的工程菌 ,表达的SARS病毒的融合蛋白约占菌体蛋白总量的 30 %左右 ,部分以可溶性形式存在。经离子交换柱和反相高压液相纯化获得了表达的融合蛋白 ,经初步鉴定 ,显示该融合蛋白有较好的抗原性和特异性.     

丙肝抗原决定簇与CTB的融合、表达及融合蛋白的纯化

通过固相化学合成法合成了编码丙型肝炎病毒(HCV)结构区和非结构区的4个抗原决定簇基因,这些抗原决定簇基因片段以不同方式串联后与ctxB基因融合,构建了12种表达不同嵌合蛋白的重组质粒,各重组质粒转化大肠杆菌后均能高效分泌性表达融合蛋白,表达产量在10～50μg/ml之间,随所融合的抗原决定簇不同而不同,表达水平主要与抗原决定簇的氨基酸组成有关,而与抗原决定簇的大小及串联次数关系不大。融合蛋白通过亲和层析纯化,达到了电泳纯,为进一步研究融合蛋白的抗原性及用作抗-HCV ELISA诊断试剂抗原打下了基础。     

丙型肝炎病毒抗原决定簇与霍乱毒素B亚基的融合表达以及融合蛋白的抗原性

通过固相化学合成法,合成了编码丙型肝炎病毒（ＨＣＶ）结构区和非结构区的４个抗原决定簇基因,这些抗原决定簇基因片段以不同方式串朕后与ｃｔｘＢ基因融合,构建了１２种表达不同嵌合蛋白的重组质粒。各重组质粒转化大肠杆菌后均能高效分泌性表达融合蛋白,表达产量随所融合的抗原决定簇不同在１０～５０μｇ／ｍｌ之间,表达水平主要与抗原决定簇的氨基酸组成有关,而与抗原决定簇的大小及串联次数关系不大。融合蛋白通过亲和层析纯化达到了电泳纯。对１１种融合蛋白中ＨＣＶ抗原决定簇的反应原性的研究表明,多数融合蛋白中ＨＣＶ抗原决定簇均能与对应的ＨＣＶ抗体结合。其中以融合蛋白９５０８２为抗原研制的抗－ＨＣＶＥＬＩＳＡ试剂具有良好的灵敏度和特异性。     

结核分枝杆菌ESXB-ESXA融合蛋白的表达及其作为检测抗原的初步应用

目的:探索一种大量表达结核分枝杆菌ESXB-ESXA融合蛋白的方法,分析其抗原性,评价其在结核抗体检测中的初步应用。方法:采用PCR方法从结核分枝杆菌基因组中分别扩增esxB和esxA基因,用Linker(G4S)3连接2条目的片段,连接pET-32a(+)载体,构建ESXB-ESXA融合蛋白表达载体;重组表达载体转化宿主细胞大肠杆菌BL21(DE3),IPTG诱导表达、纯化,Western印迹分析重组蛋白的抗原性;建立以融合重组蛋白为抗原的ELISA和胶体金检测方法,分析其在结核病抗体检测中的应用。结果:构建了ESXB-ESXA融合蛋白的表达载体,重组ESXB-ESXA在大肠杆菌中得以高效表达,表达量占菌体总蛋白的70%以上;Western印迹分析表明该重组蛋白具有较好的抗原性;ELISA和胶体金检测结果显示,用重组ESXB-ESXA抗原检测临床结核病患者有较好的特异性。结论:重组ESXB-ESXA融合蛋白表达量高并具有较好的抗原性,可作为结核病抗体检测的备选抗原。     

人巨细胞病毒gp52蛋白与pp65蛋白融合表达及初步应用

建立以重组表达HCMV蛋白为抗原的检测试剂盒,提高试剂盒的敏感性及特异性。从病毒及质粒中分别扩增gp52（281～433aa,f1）及pp65（361～473aa,f2）2个片段,以不同酶切位点同时插入到pTrcHisB载体上,筛选正确的重组质粒并在大肠杆菌中诱导表达。表达蛋白rp52～65占菌体总蛋白的30％,以包涵体的形式存在,分子量为35000.以金属螯合亲和层析（IMAC）及分子筛方法纯化表达蛋白,纯度达到96％,得率为22．9％。以间接法检测HCMVIgM阳性血清,敏感性达到90．4％（19／21）。融合蛋白具有良好的抗原性,可代替gp52及pp65作为HCMVIgM抗体检测试剂盒的包被抗原。     

HIV-1 Gag多表位嵌合基因的构建及表达蛋白的抗体制备

旨在通过构建Gag的抗原多表位融合基因及在原核系统的高表达,为HIV诊断及可能的疫苗制备提供试验基础。选定HIV-1 Gag基因中3个片段包含较多抗原表位的区域,设计带有酶切位点的引物,用PCR的方法从HIV-1HXB2全基因扩增编码这3个片段的基因序列,通过质粒提取、酶切、测序方法鉴定基因片段的正确性,SDS-PAGE和Western blotting测定融合蛋白的表达,并免疫动物制备相应抗体。结果显示,构建的HIV-1 Gag多表位嵌合基因的原核表达质粒,酶切和测序结果表明基因序列正确,基因全长576bp。在大肠杆菌BL21(DE3)中高效表达的重组蛋白分子量为27kD,以包涵体的形式存在。纯化目的蛋白免疫家兔,制备多克隆抗体IgG。ELISA和免疫荧光方法检测显示制备的多克隆抗体能具有特异性反应。成功构建和高表达了HIV-1 Gag多表位融合蛋白,纯化蛋白制备的抗体与HIV-1Gag有特异性结合。为进一步研究HIV-1奠定了试验基础。     

丙型肝炎病毒抗原决定簇与霍乱毒素B亚基的融合表达以及融合蛋白的抗…

通过固相化学合成法,合成了编码丙型肝炎病毒（ＨＣＶ）结构区和非结构区的４个抗原决定簇基因。这些抗原决定簇基因片段以不同方式串联后与ｃｔｘＢ基因融合,构建了１２种表达不同嵌合蛋白的重组质粒。各重组质粒转化大肠杆菌后均能高效分泌性表达融合蛋白,表达产量随所融合的抗原簇不同在１０－５０μｇ／ｍｌ之间,表达水平主要与抗原决定簇的氨基酸组成有关,而与抗原决定簇的大小及串联次数关系不大。融合蛋白通过亲和层析纯     

猪丹毒丝菌C43065株表面保护性抗原AN端保护区在大肠杆菌中的表达

目的：在大肠杆菌中表达猪丹毒丝菌C43065株表面保护性抗原A（SpaA）N端保护区（SpaA-N）,并检测其抗原性。方法：利用PCR方法从猪丹毒丝菌C43065株基因组中扩增出spaA基因片段,构建pMD18-spaA重组质粒并对插入片段进行测序;以pMD18-spaA重组质粒为模板,PCR扩增得到spaA-N基因片段,构建重组表达质粒pGEX-spaA-N,经序列测定证实正确后转化大肠杆菌BL21（DE3）,再经IPTG诱导表达GST-SpaA-N融合蛋白并纯化。结果：扩增得到的spaA基因长1881bp,编码由626个氨基酸残基构成的多肽;SDS-PAGE和Western印迹检测结果表明,诱导表达获得相对分子质量约64000的GST-SpaA-N融合蛋白,该融合蛋白能与相应抗体发生特异性反应。结论：获得了在大肠杆菌中可溶性表达的GST-SpaA-N融合蛋白,为进一步研究猪丹毒丝菌免疫保护性抗原奠定了基础。     

EB病毒早期蛋白P54的原核表达及其免疫原性的研究

PCR法获得编码EB病毒早期蛋白P54的基因BMRFl,序列分析后亚克隆入原核表达载体pET30a。表达质粒pET30a-BMRF1在大肠杆菌BL21(DE3)菌株中经IPTG诱导后表达了P54抗原,SDS—PAGE表明其相对分子质量为51000;采用镍离子亲和柱纯化重组蛋白。Western印迹结果表明纯化蛋白免疫BALB／c小鼠后产生了P54特异性抗体。间接免疫荧光表明免疫血清可以识别激活的Raji细胞中表达的P54蛋白。以上结果表明构建了原核表达质粒pET30a-BMRF1并在大肠杆菌细胞中成功表达EB病毒早期蛋白P54,表达蛋白具有很好的抗原性和免疫原性。     

Toxoplasma gondii: enzyme-linked immunosorbent assay using different fragments of recombinant microneme protein 1 (MIC1) for detection of immunoglobulin G antibodies

Three different fragments of microneme 1 protein termed, r-MIC1ex2, r-MIC1ex34 and r-MIC1 of Toxoplasma gondii, were expressed in Escherichia coli as fusion proteins containing six histidyl residues at N- and C-terminal. After purification by metal affinity chromatography, these recombinant proteins were tested for their usefulness as antigens in an enzyme-linked immunosorbent assay for the detection of immunoglobulin G. Ninety-eight sera from patients with different stages of invasion and 24 sera from seronegative patients were examined. There was no significant difference observed in the antigenicity for human serum samples from patients with acute toxoplasmosis between three recombinant types of MIC1 antigen (96.1% for r-MIC1ex2 antigen and 100% for both r-MIC1ex34 and r-MIC1 proteins). Sera from chronic infections (with low titers of IgG antibody) showed significant lower sensitivity, especially for r-MIC1ex34 and r-MIC1 antigens (75%, 52.7% and 36.1% for r-MIC1ex2, r-MIC1ex34 and r-MIC1, respectively). These results indicate that the strongest antigenic region of the MIC1 is encoding by the second exon of mic1 gene. When r-MIC1ex2 (N-terminal fragment of protein) was combined with MAG1 (matrix antigen) and MIC3 (microneme 3 protein), the sensitivity increased to 88.9%. This result was comparable to an ELISA using a Toxoplasma lysate antigen (TLA) and two combinations of recombinant antigens: M1 (GRA1+GRA7+SAG1) and M2 (P35+SAG2+GRA6) with the sensitivity for serum samples tested 94.4%, 88.9% and 94.4%, respectively.     

Production and Evaluation of Toxoplasma gondii Recombinant GRA7 for Serodiagnosis of Human Infections

The precise diagnosis of the acute toxoplasmosis in pregnant women and immunocompromsied patients has critical importance. Most of the commercially available assays use the whole Toxoplasma soluble extract as the antigen. However, the assays currently available for the detection of specific anti-Toxoplasma antibodies may vary in their abilities to detect serum immunoglobulins, due to the lack of a purified standardized antigen. The aim of this study was production and evaluation of the usefulness of the recombinant Toxoplasma gondii GRA7 antigen for the serodiagnosis of Toxoplasma gondii IgM and IgG by ELISA. A total of 70 T. gondii IgM positive sera, 74 T. gondii IgG positive sera, and 60 sera from subjects who were not infected with T. gondii were examined. These sera were shown different absorbance values in ELISA test. To control the specificity of the rGRA7 other parasitic diseases, for example, echinococcosis, malaria, leishmaniasis, fascioliasis, and strongyloidiasis were tested of which none showed positive results. Sensitivity and specificity of the generated recombinant IgG ELISA in comparison with commercial ELISA (com ELISA) were 89% and 90%, and the sensitivity and specificity of the generated recombinant IgM ELISA were 96% and 90%, respectively. The results obtained here show that this antigen is useful for diagnostic purposes.     

Toxoplasma gondii: an evaluation of diagnostic value of recombinant antigens in a murine model

Laboratory diagnostics of toxoplasmosis depends primarily on serological methods detecting specific antibodies. Since these methods do not always enable specific and sensitive recognition of the infection and phase of toxoplasmosis, the search for new diagnostic tools continues. Recombinant antigens promise a new alternative in diagnostics of Toxoplasma gondii infections. In this work the usefulness of six recombinant T. gondii antigens: GRA1, GRA6, GRA7, p35, SAG1, and SAG2 in the detection of primary murine toxoplasmosis was evaluated. Sera obtained from infected mice differing in their natural susceptibility to T. gondii infection, BALB/c (relatively resistant) and C57BL/6 (relatively susceptible), were tested using ELISA. During acute infection high response to GRA7, GRA6, and p35 antigens was noticed, whereas a strong reactivity with surface antigens SAG1 and SAG2 was characteristic for chronic toxoplasmosis. Our results show that the recombinant antigens are useful in distinguishing between acute and chronic toxoplasmosis regardless of the genetically determined susceptibility of the host.     

重组人巨细胞病毒(HCMV)优势表位嵌合抗原的构建表达以及捕获法检测IgM抗体

勘误：重组人巨细胞病毒(HCMV)优势表位嵌合抗原的构建表达以及捕获法检测 IgM 抗体     

Molecular typing of Toxoplasma gondii strains by GRA6 gene sequence analysis

The utility of sequence polymorphisms in the dense granule antigen GRA6 gene as typing markers for Toxoplasma gondii was investigated. The coding region of GRA6 was amplified, sequenced and compared for 30 Toxoplasma strains from eight different zymodemes (Z1-Z8). Sequence alignment identified nucleotide polymorphisms at 24 positions out of 690 bp, which correlated with murine-virulence. Types I, II, and III could be distinguished from each other on the basis of three, 10, and six variable positions, respectively. Two deletions of 15 bp and 3 bp existed in the avirulent (type II) strains. With one exception, all polymorphic positions resulted in amino acid substitutions, and the two gaps of 15 bp and 3 bp caused the deletion of six amino acids in type II strains. Intra-specific polymorphisms were also found in the virulent group. A high degree of sequence polymorphism correlating with the phenotypes of T. gondii strains points to the GRA6 gene being a good marker for strain characterisation and typing of the isolates of this apicomplexan. The large variety of amino acid changes supports the view that the GRA6 protein plays an important role in the antigenicity and pathogenicity of T. gondii. The existence of polymorphic restriction sites for endonuclease MseI was used to develop a PCR-RFLP method which could simply differentiate the three different groups (types I, II, III) of T. gondii.     

Development and clinical evaluation of a rapid serodiagnostic test for toxoplasmosis of cats using recombinant SAG1 antigen

Rapid serodiagnostic methods for Toxoplasma gondii infection in cats are urgently needed for effective control of transmission routes toward human infections. In this work, 4 recombinant T. gondii antigens (SAG1, SAG2, GRA3, and GRA6) were produced and tested for the development of rapid diagnostic test (RDT). The proteins were expressed in Escherichia coli, affinity-purified, and applied onto the nitrocellulose membrane of the test strip. The recombinant SAG1 (rSAG1) showed the strongest antigenic activity and highest specificity among them. We also performed clinical evaluation of the rSAG1-loaded RDT in 182 cat sera (55 household and 127 stray cats). The kit showed 0.88 of kappa value comparing with a commercialized ELISA kit, which indicated a significant correlation between rSAG1-loaded RDT and the ELISA kit. The overall sensitivity and specificity of the RDT were 100% (23/23) and 99.4% (158/159), respectively. The rSAG1-loaded RDT is rapid, easy to use, and highly accurate. Thus, it would be a suitable diagnostic tool for rapid detection of antibodies in T. gondii-infected cats under field conditions.     

弓形虫上海株的棒状体蛋白ROP2的克隆表达及纯化

根据已发表基因序列(GenBank登录号为Z36906)设计引物,以弓形虫(Toxoplasma gondii)上海本地株的基因组DNA为模板,扩增编码ROP2(rhpotry protein2)蛋白的基因片段,定向克隆至表达质粒pET32a(+),重组质粒经限制性酶切鉴定后测序,结果表明插入片段长度为1044bp,与GenBank上登录的序列相比,同源性为96%-100%,其中与弓形虫RH株的rop2基因同源性为100%。重组原核表达质粒pET32a-rop2转化至大肠杆菌BL21(DE3),经诱导可表达分子量约60.9kD的融合蛋白,能被感染弓形虫RH株的绵羊阳性血清识别。     

HCV核心蛋白在大肠杆菌中的表达及免疫学特性分析

目的:表达HCV核心蛋白,为检测丙肝病毒提供合适抗原。方法:以含HCV核心全长cDNA克隆的pMD18T/core质粒为模板,PCR扩增全长的HCV核心抗原基因,插入表达载体pQEN1构建重组质粒pQEN1/Core,转化BL-21(DE3)大肠杆菌,IPTG诱导表达6×His融合蛋白,表达产物经SDS-PAGE及Western blot检测和鉴定。结果:经SDS-PAGE及Western blot显示HCV核心蛋白在大肠杆菌中正确表达,融合蛋白分子量约为22 kD,表达量约占菌体蛋白总量的30%。纯化后的C蛋白能与慢性丙型肝炎患者有血清反应。结论:HCV核心蛋白在大肠杆菌中成功表达并具有较强的抗原性。     

Evaluation of serodiagnosis of toxoplasmosis by using the recombinant nucleoside triphosphate hydrolase isoforms expressed in Escherichia coli

The nucleoside triphosphate hydrolase (NTPase) isoforms termed, NTPase-I and NTPase-II of Toxoplasma gondii, were expressed in Escherichia coli as inclusion bodies and purified under denaturing condition. Furthermore, NTPase-I was refolded as an active form and purified under non-denaturing condition. The purified NTPase isoforms, both denatured and refolded, were tested for their usefulness as antigens for the serodiagnosis of acute toxoplasmosis in immunocompetent humans. The test was conducted by using the recombinant NTPase isoforms and comparing the enzyme linked immunosorbent assay (ELISA) absorbances with the Sabin-Feldman dye test titer. Seventy-three sera from dye test-positive patients, and 30 sera from subjects with no T. gondii infection were examined. The total positive rates in dye test positive sera were: 82% (60/73) for denatured NTPase-I; 78% (57/73) for denatured NTPase-II; and 63% (46/73) for refolded NTPase-I. For all three antigen types of recombinant NTPase, the positive rates of sera of acute toxoplasmosis suspected patients were 93% (13/14). A moderate correlation between the ELISA absorbance using these antigens and the dye test titer was observed with the correlation coefficients, 0.583 (r2) for denatured NTPase-I, 0.472 (r2) for denatured NTPase-II, and 0.604 (r2) for refolded NTPase-I in the linear regression analysis. There was no significant difference observed in the antigenicity between refolded and denatured NTPase-I, nor between the isoforms.     

Identification of a sporozoite-specific antigen from Toxoplasma gondii

Reduction of risk for human and food animal infection with Toxoplasma gondii is hampered by the lack of epidemiological data documenting the predominant routes of infection (oocyst vs. tissue cyst consumption) in horizontally transmitted toxoplasmosis. Existing serological assays can determine previous exposure to the parasite, but not the route of infection. We have used difference gel electrophoresis, in combination with tandem mass spectroscopy and Western blot, to identify a sporozoite-specific protein (T. gondii embryogenesis-related protein [TgERP]), which elicited antibody and differentiated oocyst- versus tissue cyst-induced infection in pigs and mice. The recombinant protein was selected from a cDNA library constructed from T. gondii sporozoites; this protein was used in Western blots and probed with sera from T. gondii -infected humans. Serum antibody to TgERP was detected in humans within 6-8 mo of initial oocyst-acquired infection. Of 163 individuals in the acute stage of infection (anti- T. gondii IgM detected in sera, or < 30 in the IgG avidity test), 103 (63.2%) had detectable antibodies that reacted with TgERP. Of 176 individuals with unknown infection route and in the chronic stage of infection (no anti- T. gondii IgM detected in sera, or > 30 in the IgG avidity test), antibody to TgERP was detected in 31 (17.6%). None of the 132 uninfected individuals tested had detectable antibody to TgERP. These data suggest that TgERP may be useful in detecting exposure to sporozoites in early T. gondii infection and implicates oocysts as the agent of infection.