布鲁菌外膜蛋白Omp31原核表达及抗原性分析

克隆羊布鲁菌的外膜蛋白Omp31基因,在大肠埃希菌中表达、纯化,并对Omp31蛋白的抗原性进行分析。以羊布鲁菌的染色体DNA为模板,扩增Omp31基因,双酶切后克隆至pET32a上,在大肠埃希菌ER2566(DE3)中诱导表达,组氨酸结合树脂柱纯化,Western blot鉴定Omp31蛋白的抗原性。将Omp31克隆至载体pET32a,提取的重组质粒经PCR鉴定、双酶切鉴定和测序分析确定目的基因成功插入到了克隆载体中。将重组质粒转化于大肠埃希菌ER2566(DE3)中表达获得HIS融合蛋白,SDS-PAGE分析证明,表达产物为43 ku的融合蛋白。Western blot结果表明,表达的蛋白具有与布鲁菌外膜蛋白相同的抗原性。     

布鲁氏菌Omp25的表达及抗原性分析

克隆羊外膜蛋白Omp25基因,在大肠杆菌中表达、纯化,并对Omp25蛋白的抗原性进行分析.以布鲁氏菌染色体DNA模板,扩增Omp25基因,双酶切后克隆至pET32a上,在大肠杆菌ER2566 (DE3)中诱导表达,组氨酸结合树脂柱纯化,Western blotting鉴定Omp25蛋白的免疫原性.将Omp25克隆至载体pET32a,提取的重组质粒经PCR鉴定、双酶切鉴定和测序分析确定目的基因成功插入到了克隆载体中.将重组质粒转化于大肠杆菌ER2566 (DE3)中表达获得HIS融合蛋白,SDS-PAGE分析证明,表达产物为43 000的融合蛋白.Western blotting分析表明,所表达的蛋白具有免疫原性.结果表明,成功地表达并纯化了Omp25蛋白,而且纯化的蛋白具有一定的免疫原性.本试验为进一步研究Omp25蛋白的功能以及寻找布鲁氏菌的诊断性蛋白奠定了基础.     

流产型布鲁菌Omp10、Omp25蛋白的表达纯化与鉴定

获得高纯度具有生物学活性的重组布鲁菌Omp10、Omp25融合蛋白,并进行抗原性的分析。将用PCR扩增出的布鲁菌Omp10、Omp25基因片段分别克隆到原核表达载体pET-32α中,构建pET-32α-Omp10/Omp25原核表达质粒。将其转入大肠杆菌BL21（DE3）PlysS中,用IPTG诱导表达,经HisTrap HP亲和层析柱分离纯化,分别用Western-blot和间接ELISA检测产物的抗原性。基因测序及酶切鉴定证明pET-32α-Omp10/Omp25原核表达载体构建成功。SDS-PAGE表明,Omp10、Omp25融合蛋白均以包涵体的形式在大肠杆菌中高效表达。经过包涵体的变性、复性及亲和层析纯化,成功获得了大小分别为34 000和44 000的融合蛋白,与预测的相对蛋白分子质量一致。Western和间接ELISA试验证明纯化的Omp10、Omp25融合蛋白能被免疫的牛布鲁菌阳性血清所识别。结果表明,成功获得了布鲁菌Omp10、Omp25融合蛋白,且均具有一定的免疫原性,通过血清学反应证实,Omp10、Omp25蛋白为布鲁菌病临床诊断试剂盒的研制奠定了基础。     

布鲁氏菌外膜蛋白16基因的克隆及原核表达

为了成功克隆外膜蛋白16(outer membrane proteins 16, Omp16)基因并对其进行原核表达,试验根据GenBank中羊布鲁氏菌M5-90株外膜蛋白Omp16基因序列(登录号:JF918760.1)设计1对引物,从布鲁氏菌基因组中扩增出大小约为507 bp的目的基因片段,凝胶回收纯化目的片段,连接入pMD20-T质粒,转化E.coli DH5α并测序,测序正确后再亚克隆入pET-28a(+)表达载体,构建重组质粒pET-Omp16,转化入E.coli BL21(DE3),经IPTG诱导其表达,最后用Western blotting分析方法鉴定诱导得到的蛋白。结果表明,成功构建了pET-Omp16原核表达载体,并在E.coli BL21中表达了Omp16基因,诱导得到的蛋白经鉴定与目的蛋白大小一致,证明成功表达了目的基因。     

布鲁氏菌外膜蛋白2b基因的克隆、原核表达及蛋白生物信息学分析

本试验旨在克隆布鲁氏菌外膜蛋白2b（Omp2b）基因并进行原核表达和蛋白的生物信息学分析。根据布鲁氏菌M5-90株外膜蛋白Omp2b基因序列设计引物,以布鲁氏菌基因组为模板,通过PCR技术扩增得到Omp2b基因片段,回收纯化后,将此片段连接入pMD20-T质粒,将该重组质粒转化E.coli DH5α感受态细胞,挑取阳性克隆菌提取质粒后,送公司测序。将该片段亚克隆入pET28a载体,构建pET28a-Omp2b表达载体,转化E.coli BL21（DE3）菌株,IPTG诱导其表达,用SDS-PAGE和Western blotting分析鉴定此蛋白。运用DNAMAN、BioEdit等各种工具软件对Omp2b基因编码的氨基酸序列进行分析。结果显示,成功克隆了Omp2b基因,其开放阅读框为1041 bp,编码347个氨基酸;构建了pET28a-Omp2b原核表达载体,并在E.coli BL21（DE3）中成功表达了Omp2b基因,表达蛋白约38 ku;Omp2b蛋白二级结构中α-螺旋、伸展链、β-折叠和无规卷曲分别占20.17%、26.22%、5.76%和47.84%。     

羊种布鲁氏菌Omp10基因的克隆及原核表达

根据GenBank公布的羊布鲁氏菌(B.melitensis) M5-90株外膜蛋白(outer membrane protein,Omp)基因序列,设计1对引物,以其全基因组为模板,采用PCR技术对其进行扩增,得到381 bp的目的片段,连接入pMD20-T载体,转化E.coli DH5α感受态细胞;测序正确后,构建pET-28a-Omp10原核表达质粒,再将该质粒转化入E.coli BL21(DE3), IPTG诱导表达融合蛋白His-Omp10,用SDS-PAGE和Western blotting进行分析.结果表明, 成功构建了含Omp10基因的原核表达载体,并在E.coli BL21(DE3)中表达了Omp10基因,诱导得到的融合蛋白经鉴定与目的蛋白大小一致,证明Omp10得到成功表达.该试验为布鲁氏菌病的进一步研究奠定基础.     

布鲁菌外膜蛋白Omp22基因的原核表达与生物信息学分析

克隆布鲁菌Omp22基因,原核表达后进行生物学信息分析。根据GenBank中羊种布鲁菌M5-90株基因组PCR扩增出639bp的目的基因片段,构建克隆重组质粒pMD-20T-Omp22,转化入E.coli DH5α。测序正确后构建表达重组质粒pET-28a-Omp22,转化入E.coli BL21(DE3)。IPTG诱导表达,Western blot鉴定诱导融合蛋白。结果表明,成功克隆Omp22基因,构建pET-28a-Omp22原核表达载体,在E.coli BL21(DE3)中表达Omp22基因。DNA Man和BIOEDIT软件生物性息学分析Omp22融合蛋白二级结构中α-螺旋占22.17%;伸展链占19.81%;β-折叠占2.83%;无规卷曲占55.19%。说明该蛋白具有较高亲水性。     

新西兰兔SOD1基因的分泌型原核表达载体构建及在E.coli表达

为了研究新西兰兔SOD1蛋白分子的结构与功能,设计了一对特异性引物,通过RT-PCR方法克隆得到了该基因编码序列。将该编码序列克隆到分泌型原核表达载体PET-25b,经菌落PCR、双酶切和测序鉴定证实成功构建了重组表达载体。将重组质粒转化大肠杆菌表达菌株E.coli BL21(de3) PlysS,建立了分泌型原核表达系统。IPTG诱导培养重组菌后收集样品检测,经SDS-PAGE、Western blot鉴定,证实在培养物上清中检测到了SOD1蛋白的表达。至此,成功建立了新西兰兔SOD1基因的分泌型原核表达系统,实现了蛋白的可溶表达,为进一步研究该蛋白的结构与功能打下了基础。     

单增李斯特菌lmo2193基因克隆与原核表达

为了对单增李斯特菌新疆绵羊脑炎临床分离株LM90SB2的lmo2193基因进行克隆及其原核表达,采用PCR方法扩增lmo2193基因,连接pMD19-T载体进行克隆,筛选阳性菌进行测序比对。将目的基因克隆至原核表达质粒p ET32a中,构建重组质粒pET32a-2193,并转化大肠杆菌感受态细胞,经诱导表达后,利用SDS-PAGE和Western blot鉴定重组蛋白。结果显示:扩增得到的lmo2193基因序列长度为1 077 bp,与预期一致;该基因在大肠杆菌中大量表达,经SDS-PAGE检测和Western blot鉴定分析表明该产物为1个60 ku左右的融合重组蛋白。本研究成功克隆lmo2193基因,并获得大量表达,为进一步研究lmo2193基因功能奠定基础。     

猪EP1基因的克隆与原核表达

为进一步体外研究EP1基因的生物学功能,本试验对EP1基因进行了组织分布分析。以猪骨髓cDNA为模板克隆了猪EP1基因的开放阅读框,对克隆的基因序列进行了序列分析,用非酶连接技术将此基因克隆至丙酸诱导型原核表达载体pBbB3a-His6-MBP-LIC中。用菌液PCR进行阳性克隆鉴定并测序,将测序鉴定正确的克隆菌液提取质粒,转化至E.coli BL21(DE3)中。丙酸钠诱导表达His6-MBP-EP1融合蛋白,并用Western blot进行鉴定。结果显示:本试验成功克隆了猪EP1基因,长度为651bp,猪EP1基因在骨髓中的表达量很高;构建了EP1丙酸诱导型原核表达载体pBbB3a-His6-MBP-EP1;His6-MBP-EP1融合蛋白在裂解菌液的上清中表达,相对分子质量为65560。结果表明,运用大肠杆菌表达系统成功表达EP1基因融合蛋白,为进一步在体外开展猪EP1基因生物学功能的研究提供基础。     

布鲁菌外膜蛋白基因Omp22克隆测序及生物信息学分析

根据GenBank中的布鲁菌属特异性基因序列Omp22设计1对引物,以牛布鲁菌（Brucella）通辽市分离菌株染色体DNA作为模板,进行PCR扩增。将其定向插入克隆载体PGEM-7Zf,综合利用生物信息学软件（Danman和Vector NTI）、数据库（Prosite和PDB）和网络服务器对外膜蛋白Omp22进行基本性质分析、三维结构预测和功能预测。结果显示,用PCR方法扩增的Omp22基因长度为639bp,编码212个氨基酸,预测其蛋白质的相对分子质量约为22 000,同时用于预测B细胞线性表位的柔韧性和亲水性参数得到综合阐释,生物信息学预测和分析结果可为研究在布氏菌致病及机体免疫应答中外膜蛋白Omp22的结构与功能的关系提供丰富资料。     

Omp25蛋白的真核表达及其对巨噬细胞的作用

本试验旨在构建表达猪布鲁菌（Brucellasuis）外膜蛋白0mp25蛋白的真核重组表达质粒,并探讨Omp25蛋白对猪肺泡巨噬细胞生长活性及细胞因子分泌的影响。以猪布鲁菌基因组DNA为模板,设计带有6×His标签的引物克隆Orap25基因,扩增产物克隆入pCI-neo真核表达载体,双酶切及测序鉴定正确后转染猪肺泡巨噬细胞3D4／21,检测其对巨噬细胞生长活性和分泌细胞因子的影响。RT—PCR和Westernblotting检测结果证实Omp25蛋白获得正确表达。显微镜观察细胞形态表明,Omp25蛋白对猪肺泡巨噬细胞的形态无明显影响。MTT结果表明,Orap25蛋白对猪肺泡巨噬细胞的生长活性有轻度但不显著的抑制作用。ELISA检测TNF—α和IL-12蛋白表达水平表明,Omp25蛋白可以显著抑制巨噬细胞分泌TNF-α和IL-12。本试验成功构建了表达猪布鲁菌外膜蛋白Omp25蛋白的真核重组表达质粒,该蛋白对猪肺泡巨噬细胞的细胞形态没有明显影响,对其生长活性有轻度但不显著的抑制作用,可以显著抑制巨噬细胞分泌TNF—α和IL-12。     

布鲁氏菌omp28基因的克隆、表达及反应原性分析

本研究克隆了羊种布鲁氏菌16M株、羊种布鲁氏菌M28株、犬种布鲁氏菌、绵羊附睾种布鲁氏菌、牛种布鲁氏菌A19株、猪种布鲁氏菌S2株的omp28基因并对以上不同种菌株的omp28基因序列及编码的氨基酸序列进行了比对,结果显示不同种布鲁氏菌omp28基因之间仅6个碱基不同,而且只有2个氨基酸不同,亲水性分析结果显示两处氨基酸的差异对蛋白亲水性不造成影响.将羊种布鲁氏菌16M的omp28基因亚克隆到pET32a中表达,OMP28在低温下诱导以可溶性形式高效表达.Westem-blot结果显示OMP28反应原性良好,是布鲁氏菌病诊断抗原的可能选择.     

Immunogenicity of recombinant Omp31 from Brucella melitensis in rams and serum bactericidal activity against B. ovis

Detergent-extracted recombinant Omp31 (rOmp31 extract) from Brucella melitensis produced in Escherichia coli was previously identified as a protective immunogen against B. ovis in mice. In this study, we evaluated the immunogenicity of rOmp31extract in rams. This immunogen was emulsified in an oil adjuvant and administered three times with 4 and 8 weeks intervals. Antibody response was measured in serum by whole B. ovis ELISA. Specific antibodies to purified rOmp31 (pET-Omp31) were detected by Western blotting and indirect ELISA. In addition, isotype specific antibodies were measured in tears. Serum bactericidal activity against B. ovis in the presence of complement was measured in vitro. Cellular immune response was explored by intradermal testing with purified rOmp31. Immunization with rOmp31 extract induced IgG specific antibodies in serum able to bind to whole B. ovis cells. Furthermore, strong inhibition in a competitive ELISA (with an Omp31-specific monoclonal antibody) suggested that a proportion of Omp31-specific antibodies were directed against a loop containing a protective epitope. Serum antibodies killed efficiently B. ovis in vitro in the presence of either guinea pig or ovine serum. Tears had both IgG and IgA antibodies to equivalent titers. Finally, immunized rams showed skin reactivity to Omp31. These data demonstrate that B. melitensis Omp31, a protective antigen identified in the mouse model, induces antibody and cellular immune mechanisms in sheep.     

Molecular characterization of Babesia canis canis isolates from naturally infected dogs in Poland

Babesia canis has generally been considered the only large Babesia to infect dogs. In this study, we used PCR to detect and characterize B. canis canis isolated from naturally infected dogs in Poland by amplifying and sequencing a portion of the 18S ribosomal RNA (rRNA) gene. Venous blood samples were collected from 76 Babesia-symptomatic dogs. A 559-bp fragment of the B. canis canis 18S rRNA gene was amplified by PCR. The PCR products were then digested with HincII restriction enzyme, and isolates were classified according to whether they were cut (group A) or not (group B) by this endonuclease. Sequencing of the PCR products from the isolates led to the identification of seven sequence variants (four in group A, and three in group B). Sequences were compared with GenBank sequences, and alignments showed that all B. canis canis isolates from Europe may be classified into groups A or B as defined in our study.     

Comparison and phylogenetic analysis of the heat shock protein 70 gene of Babesia parasites from dogs

The heat shock protein 70 (hsp70) genes of Babesia gibsoni, B. canis canis, B. canis vogeli, and B. canis rossi isolated from infected dogs were cloned by polymerase chain reaction (PCR) and sequenced. In the nucleotide sequence and the predicted amino acid sequence of the gene, the parasites were very similar to each other. The nucleotide sequences of the hsp70 gene had more variety than those of 18S nuclear subunit ribosomal DNA (18S rDNA). A phylogenetic analysis of these sequences and comparisons with sequences from other Babesia and Theileria species revealed that all canine babesial isolates analyzed in the present study were closely related to each other and formed one cluster. Additionally, a phylogenetic analysis of Babesia and Theileria species showed that these parasites could be divided into three groups: group A including canine babesial isolates, B. divergens, B. odocoilei, B. bovis, B. caballi, and B. ovis; group B including Theileria annulata, T. orientalis, and T. cervi; and group C including B. microti and B. rodhaini. These results suggested that a phylogenetic analysis of the hsp70 gene sequence might be helpful in classifying Babesia and Theileria species, and that canine babesial isolates might be closely related to each other, indicating their evolution from the same ancestry.     

犬布氏杆菌Cu/Zn-SOD基因的原核表达及多克隆抗体的制备

为检测犬布氏杆菌(Brucella canis)重组Cu/Zn-SOD蛋白的抗原性,本研究通过PCR方法扩增B.canis的Cu/Zn-SOD基因,将其克隆于表达载体pET-28a(+)中,构建重组表达质粒pET-SOD.将该质粒转化受体菌E.coli BL21 (DE3),经IPTG诱导后,SDS-PAGE电泳分析,...