羊种布鲁氏菌dhbC基因的克隆、原核表达及生物信息学分析

试验旨在对羊种布鲁氏菌dhbC基因进行克隆及原核表达,并对其表达蛋白进行生物信息学分析。参照GenBank中布鲁氏菌M5-90株dhbC基因序列信息设计1对引物,通过PCR反应扩增获得dhbC基因片段。将得到的dhbC基因连接到pMD20-T载体,构建pMD20-T-dhbC重组质粒并转化大肠杆菌（E.coli） DH5α感受态细胞,提取质粒进行酶切鉴定。鉴定正确后构建pET28a-dhbC重组质粒,转化E.coli BL21（DE3）感受态细胞。经IPTG诱导表达,表达产物用SDS-PAGE和Western blotting进行分析。运用生物信息学软件DNAMAN及相关在线网站ProtParam、SOPMA及Protscale对dhbC基因编码的氨基酸序列进行生物信息学分析。结果表明,试验成功克隆了大小约为1 093 bp的dhbC基因并进行了蛋白表达,表达的融合蛋白大小约为47 ku,且主要以包涵体形式存在。dhbC蛋白的分子式为C₁₈₆₆H₂₉₆₈N₅₄₄O₅₆₂S₁₅,分子质量为42 496.3 u,理论等电点（pI）为5.81,消光系数为33 835,不稳定系数为36.76,疏水指数为86.19,总平均疏水性（GRAVY）为-0.215。预测在哺乳动物网织红细胞的半衰期为30 h,其二级结构以α-螺旋（41.94%）和无规则卷曲（31.46%）为主。     

羊种布鲁氏菌LpxB基因的克隆、原核表达及其蛋白的生物信息学分析

试验旨在克隆羊种布鲁氏菌LpxB基因并进行原核表达和蛋白的生物信息学分析。以布鲁氏菌M5-90株基因组为模板,参照GenBank中M5-90株基因组DNA序列,用DNAMAN软件设计1对引物,通过聚合酶链式反应（PCR）扩增得到大小为1 188 bp的LpxB基因,将其连接入pMD20-T载体上,构建pMD20-T-LpxB重组质粒,将其转化到E.coli DH5α感受态细胞中,经BamH Ⅰ和 Xho Ⅰ双酶切鉴定正确后扩大培养。将BamH Ⅰ和 Xho Ⅰ双酶切获得的LpxB片段连接入pET-28a,构建重组质粒pET-28a-LpxB,转化到E.coli BL21（DE3）中,双酶切鉴定正确后扩大培养。经IPTG诱导其表达,用SDS-PAGE和Western blotting对蛋白进行鉴定。运用DNAMAN、BioEdit等软件对LpxB基因编码的氨基酸序列进行分析。结果表明,本研究成功克隆了LpxB基因并进行了蛋白表达,在LpxB蛋白二级结构中,α-螺旋、伸展链、β-折叠和无规卷曲分别占52.41%、14.94%、8.10%和24.55%。     

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。     

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.     

新疆绵羊布鲁氏菌外膜蛋白OMP2b的克隆与表达

表达新疆绵羊布鲁氏菌外膜抗原蛋白OMP3b的表达蛋白OMP2b，探索其作为诊断抗原和亚单位疫苗的可能性。采用PCR方法，从新疆绵羊布鲁氏菌基因组DNA中扩增出omp2b基因片段，将该片段克隆于原核表达载体PET-28a（＋），构建成重组质粒，经IPTG诱导，SDS-PAGE检测。结果表明，获得长约1083bp的PCR片段，序列分析结果与已知绵羊布鲁氏菌外膜蛋白OMP2b同源性达89.72％；SDS-PAGE检测表达产物，在相对分子质量39ku处有表达带。获得了新疆绵羊布鲁氏菌外膜蛋白OMP36的表达蛋白omp2b基因片段，并在大肠杆菌中实现了表达。     

布鲁氏菌eryA基因的克隆、原核表达及生物信息学分析

为分析羊种布鲁氏菌Rev.I株的基因结构、功能及作为诊断抗原的可能性,从Rev.I株基因组中扩增eryA基因片段,构建重组质粒pET-30a-eryA并进行原核表达、Western-blot检测,同时对该基因进行生物信息学分析。结果显示:本试验成功克隆并表达了eryA基因;经Western-blot检测发现,该基因编码蛋白可与阳性血清发生特异性反应,具有良好的免疫原性;生物信息学分析显示,该蛋白没有跨膜区结构,无信号肽,二级结构中α-螺旋比重最大;抗原表位分析显示:该蛋白含有较多的抗原决定簇。因此,推测eryA蛋白有望作为布鲁氏菌的免疫诊断抗原,这为进一步探索布鲁氏菌基因工程疫苗的研制及iELISA诊断试剂盒的建立奠定了基础。     

布鲁氏菌S19毒力基因BvfA的原核表达及生物信息学分析

试验旨在对布鲁氏菌S19毒力因子A（BvfA）基因进行克隆及原核表达,并对其编码的BvfA蛋白结构进行生物信息学分析。登录GenBank下载布鲁氏菌S19的1号染色体（登录号：CP030751.1）,根据文献报道的布鲁氏菌S19 BvfA基因的位置和BvfA蛋白的分子质量,在NCBI的ORF Finder中预测编码BvfA蛋白的开放阅读框（ORF）,根据ORF预测的BvfA基因设计扩增BvfA基因的引物,克隆该序列并连接到表达载体pET-32a（+）上,构建重组质粒pET-32a（+）-BvfA,并诱导其在大肠杆菌BL21（Ril）感受态细胞中表达,表达产物经SDS-PAGE和Western blotting验证,并用生物信息学方法对BvfA蛋白结构进行在线预测。结果显示,BvfA基因ORF全长为336 bp,表达纯化得到分子质量为11 ku的分泌型BvfA蛋白;生物信息学分析显示,BvfA蛋白属于不稳定的亲水性蛋白,无跨膜结构,第1-28位氨基酸处存在信号肽区域,BvfA蛋白55.6%定位于细胞外,有12个可能的磷酸化位点,无O-糖基化位点,BvfA蛋白二级结构主要由α-螺旋和无规则卷曲组成。以上结果可为后续研究BvfA蛋白在布鲁氏菌中的致病机制及寻找布鲁氏菌病新的治疗靶点提供一定的参考。     

新疆绵羊种布鲁氏菌外膜蛋白OMP36的表达蛋白OMP2b基因的克隆与表达

以表达新疆绵羊种布鲁氏菌外膜抗原蛋白OMP36的表达蛋白OMP2b,探索其作为诊断抗原和分子疫苗的可能性。采用PCR扩增技术,从新疆绵羊种布鲁氏菌基因组DNA中扩增出OMP2b基因片段,将该片段克隆于原核表达载体PE-28a(+),构建成重组质粒,IPTG诱导表达,SDS-PAGE检测有无蛋白的表达。结果表明,获得长约1 083bp的PCR片段,序列分析结果与已知绵羊种OMP2b同源性达89.72%;SDS-PAGE检测表达产物,在相对分子量39 ku获得了新疆绵羊种布鲁氏菌外膜蛋白OMP36的表达蛋白OMP2b基因片段,并在大肠杆菌中实现了表达。     

新疆绵羊种布鲁氏菌外膜蛋白omp2b基因的克隆与序列分析

根据已报道的绵羊种布鲁氏茵外膜蛋白基因omp2b的核酸序列设计引物，从新疆绵羊种布鲁氏菌基因组中扩增到了omp2b基因。将该片段克隆到PBS—T载体上，并对所得到的重组质粒进行酶切分析、PCR鉴定，证明所得到的片段为阳性重组子。与报道的绵羊种布鲁氏菌omp2b序列有89．72％的同源性，并在N端存在高度保守区。至此得到omp2b基因的全长克隆，通过对所克隆的基因和其他布鲁氏菌外膜蛋白omp2b的进化树分析，发现其和牛种布鲁氏茵的亲缘关系最近，同源性较高。     

羊种布鲁氏菌Wzt基因的克隆及原核表达

为进一步研究Wzt蛋白在光滑型脂多糖合成路径中的作用,本试验利用PCR技术,以羊种布鲁氏菌16 M株基因组为模板,扩增出大小为759 bp的Wzt基因片段,将其连入pMD20-T载体,测序正确后构建重组质粒pET-28a-Wzt,转化E.coli BL21(DE3)工程菌,IPTG诱导其表达,最后用Western blotting鉴定蛋白。结果显示,扩增出的Wzt基因片段大小为759 bp,与GenBank中登录的羊种布鲁氏菌16 M株Wzt基因序列(登录号:AF047478.1)同源性为99.87%,证明成功克隆了Wzt基因,同时成功构建了pET-28a-Wzt原核表达载体,并在E.coli BL21(DE3)工程菌中表达了Wzt蛋白,诱导得到的融合蛋白大小约为30 ku,位于25～35 ku之间,与目的蛋白大小一致,结果表明成功表达了目的基因。     

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。     

Cloning,Prokaryotic Expression and Bioinformatics Analysis of LpxB Gene of Brucella melitensis

The study was aimed to clone and express LpxB gene,and perform the bioinformatics analysis of protein.The genomic DNA of Brucella melitensis M5-90 was used as template.According to the genome sequence of M5-90 on GenBank,a pair of primers was designed.LpxB gene,which was 1 188 bp,was amplified by PCR,and was ligated into pMD20-T vector.The constructed recombinant plasmid pMD20-T-LpxB was transformed into E.coli DH5α.The recombinant plasmid was confirmed by endonuclease digestion and sequencing.The coding region of LpxB from pMD20-T was digested by BamHⅠ and XhoⅠ.Then,the fragment was inserted into prokaryotic expression vector pET-28a,and the positive plasmid was named pET-28a-LpxB.The pET-28a-LpxB was transformed into E.coli BL21 (DE3).The expressed protein was identified by SDS-PAGE and Western blotting.DNAMAN and BioEdit softwares were used to analyze the sequence of amino acids encoded LpxB gene.The results showed that the CDS of LpxB was successfully cloned and expressed.The secondary structure of LpxB protein consisted structure α -helix,extended strand,β-turn and random coil which accounted for 52.41％,14.94％,8.10％ and 24.55％,respectively.     

布鲁氏菌Omp19基因的生物信息学分析、克隆表达和ELISA方法的初步建立

为了进一步研究布鲁氏菌外膜蛋白19（outer membrane protein 19,OMP19）的结构与功能,并获得具有反应原性的OMP19重组蛋白,建立布鲁氏菌间接ELISA抗体检测方法,试验通过生物信息学软件对OMP19蛋白进行氨基酸序列分析,经PCR技术克隆Omp19基因,利用无缝克隆技术构建重组表达载体pET-32a-Omp19,将其转化大肠杆菌BL21（DE3）感受态细胞,诱导其表达并纯化,并通过Western blotting和ELISA分析方法分别检测OMP19蛋白的反应原性,以建立基于该蛋白的间接ELISA方法。结果显示,OMP19蛋白N端有19个信号肽序列,二级结构以无规则卷曲为主,且OMP19蛋白含有优势抗原表位,利用PCR技术和无缝克隆技术成功构建了Omp19基因的原核表达载体pET-32a-Omp19,成功诱导表达并纯化了OMP19融合蛋白,大小约为35 ku,与理论值相符,并通过Western blotting和ELISA方法鉴定OMP19的反应原性,结果表明该蛋白具有较好的反应原性,且包被浓度为10 μg/mL,一抗稀释比为1：50,二抗稀释比为1：8 000时,P/N值最大,为2.80。本研究结果为布鲁氏菌病快速诊断方法的建立和新型疫苗的研发奠定了理论基础。     

Bioinformatics Analysis,Cloning and Expression of Omp19 Gene of Brucella and Preliminary Establishment of ELISA Method

This study was aimed to further investigate the structure and function of Brucella outer membrane protein 19 (OMP19),obtain the recombinant protein of OMP19 with reactivity,and establish the new method for diagnosing brucellosis based on indirect ELISA.The amino acid sequence of OMP19 protein was analyzed by bioinformatics software,Omp19 gene was amplified by PCR,and the recombinant expression vector pET-32a-Omp19 was constructed by seamless cloning method,it was transformed into E.coli BL21 (DE3) competent cells,induced and purified OMP19 protein.The immunogenicity of OMP19 protein was detected by Western blotting and ELISA,respectively,to establish an indirect ELISA method based on the protein.The results showed that there were 19 signal peptide sequences in the N terminal of OMP19 protein,and the secondary structure was random coil,and OMP19 protein contained dominant antigenic epitopes.The prokaryotic expression vector pET-32a-Omp19 was successfully constructed by PCR and seamless cloning method,the OMP19 fusion protein was successfully induced,expressed and purified,the size of protein was about 35 ku,which was consistent with the expect size.The reactivity of OMP19 was identified by Western blotting and ELISA,the results showed that the fusion protein had good reactivity,and the coating concentration was 10 μg/mL.The dilution ratio of the first antibody was 1：50 and the second antibody dilution ratio was 1：8 000,the P/N value reach the highest at 2.80.The results laid a foundation for the establishment of rapid diagnosis of brucellosis and the development of new vaccine.     

Cloning,Prokaryotic Expression and Bioinformation Analysis of dhbC Gene of Brucella melitensis

This study was aimed to clone and express dhbC gene of Brucella melitensis, and analyze the bioinformatics of its expressed protein. A pair of primers were designed by referring to dhbC gene sequence information of Brucella melitensis M5-90 strain in GenBank, and the dhbC gene fragment was amplified by PCR method. The obtained dhbC gene was ligated into pMD20-T vector to construct pMD20-T-dhbC recombinant plasmid and transformed into E.coli DH5α competent cells. The plasmid was identified by restriction enzyme digestion. The recombinant plasmid pET28a-dhbC was constructed and transformed into E.coli BL21 (DE3) competent cells. The expression was induced by IPTG. The expressed product was analyzed by SDS-PAGE and Western blotting. Bioinformatics analysis of the amino acid sequence encoded by dhbC gene was carried out using bioinformatics software DNAMAN and related online sites ProtParam, SOPMA and Protscale. The results showed that dhbC gene was cloned with the length of 1 093 bp, and protein expression was expressed. The expressed fusion protein was about 47 ku, and was mainly in the form of inclusion body. The molecular weight of the dhbC protein was C₁₈₆₆H₂₉₆₈N₅₄₄O₅₆₂S₁₅, the molecular mass was 42 496.3 u, the theoretical isoelectric point (pI) was 5.81, the extinction coefficient was 33 835, the instability coefficient was 36.76, the hydrophobic index was 86.19, the total average hydrophobicity (GRAVY) was -0.215. The half-life of reticulocytes in mammals was predicted to be 30 h, and the secondary structure was dominated by α-helix (41.94%) and random coil (31.46%).