无标签鼠疫耶尔森菌Ⅴ抗原突变体在大肠杆菌中的表达、纯化及免疫原性分析

目的:在大肠杆菌中表达、纯化无标签鼠疫耶尔森菌低钙反应V抗原突变体,并对其免疫原性进行研究。方法:采用融合PCR方法将Ⅴ抗原基因中编码半胱氨酸的碱基缺失突变,将获得得Ⅴ抗原突变体基因克隆到原核表达载体pET32a(+)后转化大肠杆菌BL21(DE3),用IPTG诱导表达并进行柱层析纯化,纯化产物以Western印迹进行鉴定并免疫BALB/c小鼠,通过ELISA检测免疫血清效价。结果:目的蛋白在大肠杆菌中获得了可溶性高表达,经三步柱层析后纯度高于95%,经Western印迹检测可与野生型V抗原单克隆抗体特异性结合,免疫小鼠后获得高效价免疫血清。结论:获得了无标签的鼠疫耶尔森菌Ⅴ抗原突变体蛋白,并证实其具有免疫原性,将对V抗原结构和功能的研究提供帮助。     

鼠疫菌H-NS蛋白的表达与纯化及其DNA结合活性

摘要：【目的】利用大肠杆菌BL21λDE3的表达系统,表达出有活性的鼠疫耶尔森氏菌（以下简称鼠疫菌）调控子蛋白H-NS,为进一步研究H-NS的转录调控奠定基础。【方法】 PCR扩增鼠疫菌201株hns基因的编码区,将其直接克隆入pET28a质粒中,再将pET28a-hns重组质粒转入大肠杆菌BL21λDE3菌株中,所得菌株经IPTG诱导后能表达出鼠疫菌His-H-NS蛋白;通过体外的凝胶迁移实验（EMSA）和DNaseⅠ足迹实验对His-H-NS蛋白与DNA的结合活性进行分析。【结果】成功表达出有活性的鼠疫菌His-H-NS蛋白,该蛋白对鼠疫菌pH6抗原基因（psaA、psaE）及rovA基因均有结合活性。【结论】鼠疫菌His-H-NS具有DNA结合活性,说明H-NS能调控鼠疫菌基因的转录。     

重组鼠疫菌V抗原的纯化及其豚鼠免疫保护力初探

采用Ni^2 亲和层析方法,对用工程化大肠杆菌BL21(DE3)表达的重组鼠疫菌v抗原进行纯化,目标蛋白纯度达到90％以上。以氢氧化铝凝胶配制吸附疫苗,经二针次肌内注射免疫实验豚鼠后,对皮下注射400个致死剂量(MLD)强毒鼠疫菌攻击有一定保护效力,存活率为20％。结果表明,重组鼠疫菌V抗原有望作为改进的F1 V亚单位疫苗的主要成分。     

鼠疫菌PsaA 抗原的表达及抗体制备和应用

目的:制备鼠疫菌PsaA抗原及抗体,建立针对PsaA抗体的快速检测方法,并检测鼠疫感染猴血清标本中的PsaA抗体的阳性率。方法:利用PCR方法扩增出PsaA蛋白基因片段,在大肠杆菌原核表达系统中表达出重组PsaA抗原,以镍柱亲和层析纯化包涵体形式的表达蛋白,以尿素梯度透析复性成可溶蛋白。再以表达蛋白为免疫原,常规免疫家兔,收集兔血清制备多抗,并以正辛酸-硫酸铵法提纯获得PsaA抗体IgG。利用得到的PsaA抗原和抗体为材料,建立两种检测PsaA抗体的快速检测方法,即间接ELISA法和上转换发光(Up-converting Phospher Technology,UPT)免疫层析试纸条法。最后利用这两种方法检测18份猴血清标本中的PsaA抗体。结果:鼠疫菌感染猴血清标本中PsaA抗体的阳性率为62%(8/13)。结论:成功建立了针对鼠疫菌PsaA抗体的快速检测方法,并检测到13份鼠疫感染猴血清标本中的PsaA抗体的阳性率为62%。     

鼠疫菌PsaA抗原的表达及抗体制备和应用

目的：制备鼠疫菌PsaA抗原及抗体,建立针对PsaA抗体的快速检测方法,并检测鼠疫感染猴血清标本中的PsaA抗体的阳性率。方法：利用PCR方法扩增出PsaA蛋白基因片段,在大肠杆菌原核表达系统中表达出重组PsaA抗原,以镍柱亲和层析纯化包涵体形式的表达蛋白,以尿素梯度透析复性成可溶蛋白。再以表达蛋白为免疫原,常规免疫家兔,收集兔血清制备多抗,并以正辛酸-硫酸铵法提纯获得PsaA抗体IgG。利用得到的PsaA抗原和抗体为材料,建立两种检测PsaA抗体的快速检测方法,即间接ELISA法和上转换发光（Up-converting Phospher Technology,UPT）免疫层析试纸条法。最后利用这两种方法检测18份猴血清标本中的PsaA抗体。结果：鼠疫菌感染猴血清标本中PsaA抗体的阳性率为62%（8/13）。结论：成功建立了针对鼠疫菌PsaA抗体的快速检测方法,并检测到13份鼠疫感染猴血清标本中的PsaA抗体的阳性率为62%。     

无标签鼠疫耶尔森菌V抗原突变体在大肠杆菌中的表达、纯化及免疫原性分析

目的：在大肠杆菌中表达、纯化无标签鼠疫耶尔森菌低钙反应Ｖ抗原突变体,并对其免疫原性进行研究。方法：采用融合ＰＣＲ方法将Ｖ抗原基因中编码半胱氨酸的碱基缺失突变,将获得得Ｖ抗原突变体基因克隆到原核表达载体ｐＥＴ３２ａ（＋）后转化大肠杆菌ＢＬ２１（ＤＥ３）,用ＩＰＴＧ诱导表达并进行柱层析纯化,纯化产物以Ｗｅｓｔｅｒｎ印迹进行鉴定并免疫ＢＡＬＢ／ｃ小鼠,通过ＥＬＩＳＡ检测免疫血清效价。结果：目的蛋白在大肠杆菌中获得了可溶性高表达,经三步柱层析后纯度高于９５％,经Ｗｅｓｔｅｒｎ印迹检测可与野生型Ｖ抗原单克隆抗体特异性结合,免疫小鼠后获得高效价免疫血清。结论：获得了无标签的鼠疫耶尔森菌Ｖ抗原突变体蛋白,并证实其具有免疫原性,将对Ｖ抗原结构和功能的研究提供帮助。     

鼠疫菌V抗原DNA疫苗提供抗鼠疫保护

制备了在BALB／c小鼠模型中最佳化表达鼠疫菌V抗原的DNA疫苗载体。比较了6种不同真核生物启动子,选择出带有一附加转译增强子下游的CMV启动子。令人诧异的是,为在鼠细胞中的表达而采取的V抗原1crV基因密码子替换未发现能促进V抗体的应答。     

鼠疫耶尔森菌F1-V融合蛋白改构体的构建、原核表达及纯化

目的:通过基于结构的基因突变获得鼠疫耶尔森菌F1抗原突变体(F1mut),克隆、表达并纯化F1mut-V融合蛋白。方法:通过3轮PCR,将编码F1抗原分子N端1~14位氨基酸的基因序列移到3'端,测序无误后将F1mut基因与V基因的5'端连接,构建改构的融合基因F1mut-V,将其克隆到原核表达载体p ET-32a后转化大肠杆菌BL21(DE3),经IPTG诱导后,目的蛋白为可溶性表达,通过硫酸铵分级沉淀、阴离子交换层析、疏水相互作用层析和凝胶过滤层析纯化,用SDS-PAGE和Western印迹分析纯化产物。结果:重组F1mut-V在大肠杆菌中为可溶性表达,表达量占全菌蛋白的25%以上,纯化后目的蛋白的纯度达95%,经Western印迹检测,与抗V、F1抗体均有特异性结合。结论:重组F1mut-V有望成为新一代亚单位疫苗的有效成分。     

鼠疫抗原LcrV在乳酸乳球菌中的表达与鉴定

目的以乳酸乳球菌(Lactococcus lactis)为载体,将鼠疫抗原LcrV基因导入乳酸乳球菌内,构建重组肠道微生态菌株,作为黏膜免疫疫苗的先期探索和尝试。方法采用酸诱导P170启动子,乳酸乳球菌本身的SP310mut2信号肽,将鼠疫杆菌LcrV抗原结构基因克隆到质粒pAM J397上,电转化感受态Lactococcus lactis PSM565。结果经重组子PCR鉴定,SDS-PAGE检测,W estern-b lot鉴定,在Lactococcus lactisPSM565/pAM J397-V培养基上清中获得了38 kD的鼠疫抗原LcrV蛋白。结论在乳酸乳球菌中成功表达了鼠疫V抗原,为下一步鼠疫黏膜疫苗的研制打下基础。     

旋毛虫肌幼虫ES抗原的基因克隆及高效表达

作者对编码旋毛虫肌幼虫ES抗原的部分结构基因进行了克隆、鉴定和表达。用RNA PCR技术直接从旋毛虫肌幼虫总RNA中反转录并扩增出0．7kh的靶DNA,酶切分析后将其克隆到融合表达载体pEx3lC中。SDS—PAGE电泳表明,含重组子的大肠杆菌能够表达出一分子量为37kDa的融合蛋白(P37),后者占菌体总蛋白的22%以上,并以包含体形式存在于菌体中。经对纯化后表达蛋白的ELlSA检测,证明它能被猪旋毛虫病阳性血清和抗旋毛虫单克隆抗体识别。研究结果揭示,重组蛋白P37对于研制旋毛虫病诊断抗原和免疫抗原具有潜在的应用价值。     

Regulatory mechanism of simian virus 40 gene expression in permissive and in nonpermissive cells.

Primary or continuous lines of mouse cells (3T3) are nonpermissive for simian virus 40 (SV40). Abortively infected cells synthesize tumor antigen (T antigen but not viral DNA and virus capsid protein (V antigen). V antigen, however, was obtained when SV40 DNA was injected into 3T3 cells. This late gene expression also appears to be correlated with the quantity of injected DNA molecules per 3T3 cell. T antigen formation can be detected after microinjection of only 1 to 2 DNA molecules, but the intensity of intranuclear T antigen fluorescence is significantly brighter with injection of higher concentrations of viral DNA. In permissive cells (TC7), early and late SV40 gene expression is directly related to the number of injected molecules. Microinjection of 1DNA molecule induced T and V antigen formation with the same efficiency as microinjection of 2,000 to 4,000 molecules. The question of weather late SV40 gene expression is directly related to the quantity of an early virus-specific product was approached by microinjection of early SV40 complementary RNA together with small amounts of viral DNA. V antigen was obtained in a high proportion of recipient 3T3 cells at conditions where microinjection of viral DNA alone induced T but not V antigen synthesis.     

Characterization of a major protein with a molecular weight of 160,000 associated with the viral capsid of Epstein-Barr virus.

A monoclonal antibody designated V3 was produced against a late protein associated with the Epstein-Barr virus-induced viral capsid antigen complex. The antibody reacted with discrete patches in the nuclei of infected cells as well as with virus particles, as shown by immunofluorescence and ultrastructural immunoperoxidase staining. The molecular weight of the protein precipitated by this monoclonal antibody was ca. 160,000. All anti-viral capsid antigen antibody-positive sera tested in an enzyme-linked immunosorbent assay reacted with this purified protein. The synthesis of the antigen was inhibited by phosphonoacetic acid but was not affected by tunicamycin, indicating that this was a late nonglycosylated viral protein. No differences were noted between the protein isolated from the P3HR-1 and B-95-8 cell lines as determined by immunoprecipitation and peptide mapping. By isoelectric focusing, this protein had a pI on the basic side ranging from 7.5 to 9.0.     

Prevalence of a Vibrio cholerae 18-kDa antigen in vibrios

Abstract An 18-kDa protein that occurs in Vibrio cholerae has been described as an in vivo and low-iron regulated outer membrane antigen. Monoclonal antibodies which recognized this antigen were protective as passive vaccines in the infant rabbit model of cholera disease. In this study, those monoclonal antibodies were used in three immunological assays for surveillance of various bacteria for the 18-kDa antigen. ELISA, and Western blot assays gave variable results with bacteria or outer membrane preparations. The biodot assay was the most sensitive test, detecting the 18-kDa antigen in 29 of 29 V. cholerae strains, independent of biotype or serotype. A few other Gram-negative bacteria and V. parahaemolyicus reacted weakly with our antibodies and antiserum.     

Identification of two epitopes on the dengue 4 virus capsid protein recognized by a serotype-specific and a panel of serotype-cross-reactive human CD4+ cytotoxic T-lymphocyte clones.

We analyzed the CD4+ T-lymphocyte response of a donor who had received an experimental live-attenuated dengue 4 virus (D4V) vaccine. Bulk culture proliferative responses of peripheral blood mononuclear cells (PBMC) to noninfectious dengue virus (DV) antigens showed the highest proliferation to D4V antigen, with lesser, cross-reactive proliferation to D2V antigen. We established CD4+ cytotoxic T-lymphocyte clones (CTL) by stimulation with D4 antigen. Using recombinant baculovirus antigens, we identified seven CTL clones that recognized D4V capsid protein. Six of these CTL clones were cross-reactive between D2 and D4, and one clone was specific for D4. Using synthetic peptides, we found that the D4V-specific CTL clone recognized an epitope between amino acids (aa) 47 and 55 of the capsid protein, while the cross-reactive CTL clones each recognized epitopes in a separate location, between aa 83 and 92, which is conserved between D2V and D4V. This region of the capsid protein induced a variety of CD4+ T-cell responses, as indicated by the fact that six clones which recognized a peptide spanning this region showed heterogeneity in their recognition of truncations of this same peptide. The bulk culture response of the donor's PBMC to the epitope peptide spanning aa 84 to 92 was also examined. Peptides containing this epitope induced proliferation of the donor's PBMC in bulk culture, but peptides not containing the entire epitope did not induce proliferation. Also, PBMC stimulated in bulk culture with noninfectious D4V antigen lysed autologous target cells pulsed with peptides containing aa 84 to 92. These results indicate that this donor exhibits memory CD4+ T-cell responses directed against the DV capsid protein and suggest that the response to the capsid protein is dominant not only in vitro at the clonal level but in bulk culture responses as well. Since previous studies have indicated that the CTL responses to DV infection seem to be directed mainly against the envelope (E) and NS3 proteins, these results are the first to indicate that the DV capsid protein is also a target of the antiviral T-cell response.     

Optimal design features of camelized human single-domain antibody libraries

We have constructed a human V(H) library based on a camelized V(H) sequence. The library was constructed with complete randomization of 19 of the 23 CDR3 residues and was panned against two monoclonal antibody targets to generate V(H) sequences for determination of the antigen contact residue positions. Furthermore, the feasibility and desirability of introducing a disulfide bridge between CDR1 and CDR3 was investigated. Sequences derived from the library showed a bias toward the use of C-terminal CDR3 residues as antigen contact residues. Mass spectrometric analyses indicated that CDR1-CDR3 disulfide formation was universal. However, surface plasmon resonance and NMR data showed that the CDR3 constraint imposed by the disulfide bridge was not always desirable. Very high yields of soluble protein products and lack of protein aggregation, as demonstrated by the quality of the (1)H-(15)N HSQC spectra, indicated that the V(H) sequence for library construction was a good choice. These results should be useful in the design of V(H) libraries with optimal features.     

Evaluation of diagnostic efficiency of the recombinant protein modeling immunodominant epitope V3 of envelope gp120 for immunoenzyme detection for HIV-1 infection antibodies

The third hypervariable domain V3 of the human immunodeficiency virus type 1 gpl20 envelope glycoprotein contains neutralizing epitopes and plays an important role in the diagnosis of HIV infection . Neutralizing antibodies bind to conserved epitope with sequence GPG of V3 loop. The effect of sequence variation on the antigenic properties of the V3 epitope gp120 was studied using five synthetic peptides. The amino acid sequence of the peptide corresponding to the V3 region gp120 of HIV-1 subtype C showed the highest immunoreactivity. The DNA fragment encoding V3-C region gp120 was synthesized by polymerase chain reaction and cloned into pET41b vector. The recombinant plasmid was expressed in the E. coli cells, and recombinant protein was purified using glutathione-S sepharose affinity chromatography. The serological activity of the recombinant protein was tested using ELISA and compared to activity of similar synthetic peptide. The results of this study showed that most immunoreactive agent was the amino acid sequence of V3 region gp120 of HIV-1 subtype C. The recombinant antigen comprising this sequence was more antigenic than synthetic peptide with the same sequence. The evaluation of this antigen shows that this protein is a good candidate for the immunoassay development.     

Variable domain antibodies specific for viral hemorrhagic septicemia virus (VHSV) selected from a randomized IgNAR phage display library

Phage display libraries are used to screen for nucleotide sequences that encode immunoglobulin variable (V) regions that are specific for a target antigen. We previously constructed an immunoglobulin new antigen receptor (IgNAR) phage display library. Here we used this library to obtain an IgNAR V region that is specific for viral hemorrhagic septicemia virus (VHSV). A phage clone (clone 653) was found to be specific for VHSV by the biopanning method. The V region of clone 653 was used to construct a 6 × His tagged recombinant IgNAR-653 V protein (rIgNAR-653) using the Escherichia coli pET system. The rIgNAR-653 protein bound specifically to VHSV, confirming its activity.     

Strains of Vibrio cholerae serogroups O1 and O139 that produce the basic protective antigens

To find out stable and effective producers of major protective antigens intended for use as components of cholera chemical vaccine against V. cholerae strains of serogroups O and O139, the comparative analysis of the production of cholera toxin, toxin-coregulated pili (TCP), antigens O1 and O139, polysaccharide capsule and outer membrane protein OmpU in different V. cholerae strains groups O1 and O139 has been made. V. cholerae strain KM68, serogroup O1, has been found capable of the production of antigen O1, serovar Ogawa, protein OmpU at a sufficiently high level and the hyperproduction of cholera toxin and TCP, and thus suitable for use in the manufacture of cholera bivalent vaccine as the source of these antigens. Specially selected alysogenic noncapsular strain KM137 of serogroup O139, characterized by a high and stable level of the biosynthesis of this somatic antigen when grown in both laboratory and production conditions, may serve as the produces of antigen O139.     

Purification and characterization of a recombinant Yersinia pestis V-F1 "Reversed" fusion protein for use as a new subunit vaccine against plague

We previously developed a unique recombinant protein vaccine against plague composed of a fusion between the Fraction 1 capsular antigen (F1) and the V antigen. To determine if overall expression, solubility, and recovery of the F1-V fusion protein could be enhanced, we modified the original fusion. Standard recombinant DNA techniques were used to reverse the gene order such that the V antigen coding sequence was fused at its C-terminus to the N-terminus of F1. The F1 secretion signal sequence (F1S) was subsequently fused to the N-terminus of V. This new fusion protein, designated F1S-V-F1, was then co-expressed with the Y. pestis Caf1M periplasmic chaperone protein in BL21-Star Escherichia coli. Recombinant strains expressing F1-V, F1S-F1-V, or F1S-V-F1 were compared by cell fractionation, SDS-PAGE, Western blotting, and suspension immunolabelling. F1S-V-F1 exhibited enhanced solubility and secretion when co-expressed with Caf1M resulting in a recombinant protein that is processed in a similar manner to the native F1 protein. Purification of F1S-V-F1 was accomplished by anion-exchange and hydrophobic interaction chromatography. The purification method produced greater than 1mg of purified soluble protein per liter of induced culture. F1S-V-F1 polymerization characteristics were comparable to the native F1. The purified F1S-V-F1 protein appeared equivalent to F1-V in its ability to be recognized by neutralizing antibodies.