羊源类鼻疽伯克霍尔德菌BPSS1512基因克隆、原核表达及其蛋白的生物信息学分析

为克隆羊源类鼻疽伯克霍尔德菌BPSS1512基因,并对其编码的蛋白进行生物信息学分析,以类鼻疽伯克霍尔德菌基因组为模板,参照GenBank中Burkholderia pseudomallei K96243株基因组DNA序列（登录号：NC_006351.1）设计引物,PCR扩增BPSS1512基因,构建重组质粒,SDS-PAGE和Western blotting分析其蛋白表达,DNAMAN等软件对BPSS1512基因编码的氨基酸序列进行分析。结果显示,PCR扩增成功得到1 425 bp的特异性条带,BamHⅠ和Hind Ⅲ双酶切后得到约为5 000和1 500 bp的条带,表明重组质粒pET-28a-BPSS1512构建成功,IPTG浓度为10 mmol/L,诱导时间8 h为最适宜的诱导条件。BPSS1512基因编码的蛋白质分子质量为53 ku,在包涵体中表达;在BPSS1512蛋白二级结构中,α-螺旋、延伸链和无规卷曲分别占24.05%、14.77%、61.18%,并且疏水性区域分布在-2.0~+2.4之间,说明BPSS1512蛋白具有较强的疏水性,本试验结果可为类鼻疽病的防制提供参考依据。     

类鼻疽伯克霍尔德菌groEL基因的克隆、原核表达及其蛋白的生物信息学分析

试验旨在对类鼻疽伯克霍尔德菌groEL基因进行克隆与原核表达,并对其表达蛋白进行生物信息学分析。提取该菌基因组DNA作为模板,参考GenBank中类鼻疽伯克霍尔德菌groEL基因序列,设计1对引物。通过PCR扩增得到大小为1 641 bp的groEL基因片段,将其连接至pMD19-T载体,构建pMD19-T-groEL重组质粒,经BamHⅠ和Hind Ⅲ双酶切鉴定正确后,构建重组质粒pET-28a（+）-groEL。将鉴定正确的pET-28a（+）-groEL质粒转化至E.coli BL21（DE3）感受态细胞中,经IPTG诱导表达,运用SDS-PAGE和Western blotting方法进行蛋白质鉴定,利用DNAMAN和BioEdit等软件进行生物信息学分析。结果发现,试验成功克隆了类鼻疽伯克霍尔德菌groEL基因并进行了蛋白表达,表达的融合蛋白大小约为64 ku,GroEL蛋白的分子式为C₄₅₁₀H₇₃₈₁N₁₆₄₁O₁₈₄₀S₅₂₁,原子总个数为15 893,消光系数为32 500,不稳定指数为40.31,亲水性平均值为0.901。GroEL蛋白二级结构中α-螺旋（Hh）、延伸链（Ee）、无规则卷曲（Cc）分别占48.71%、13.19%和38.10%。本试验结果为深入探究类鼻疽杆菌groEL基因的分子作用机理奠定了基础。     

羊源类鼻疽伯克霍尔德菌BPSL1467基因克隆、表达及其蛋白生物信息学分析

试验旨在克隆和表达羊源性类鼻疽伯克霍尔德菌（Burkholderia pseudomallei,B.pseudomallea）的BPSL1467基因,并对其表达的蛋白进行生物信息学分析。以羊源类鼻疽伯克霍尔德菌（BPHN1株）基因组为模板,参照GenBank中类鼻疽伯克霍尔德菌K96243标准株BPSL1467基因序列设计引物,PCR扩增获得目的基因片段,将所得片段与pET-28a（+）载体连接,构建pET-28a（+）-BPSL1467重组质粒。将鉴定正确的pET-28a（+）-BPSL1467重组质粒转化大肠杆菌BL21（DE3）感受态细胞,通过IPTG诱导表达,SDS-PAGE和Western blotting鉴定表达产物。使用DNAMAN、ProtParam、SOPMA和Protscale相关生物信息学软件对BPSL1467基因编码的氨基酸序列进行分析。结果显示,本试验成功克隆了462 bp的BPSL1467基因,诱导表达重组蛋白大小约为22 ku,主要以包涵体的形式存在。BPSL1467蛋白分子式为C₇₆₃H₁₂₀₉N₂₀₃O₂₁₇S₆,分子质量为16 890.58 u;其不稳定系数为33.95,属于稳定蛋白;理论等电点（pI）为8.85,为碱性蛋白;总平均疏水性（GRAVY）为-0.190,为亲水性蛋白。该蛋白的二级结构中以无规则卷曲和α-螺旋为主。本试验结果为深入研究羊源类鼻疽伯克霍尔德菌的BPSL1467基因的分子作用机理提供了参考依据。     

Cloning,Prokaryotic Expression and Bioinformatics Analysis of BPSS0180 Gene of Burkholderia pseudomallei

This study was aimed to clone and express the BPSS0180 gene of Burkholderia pseudomallei (B. pseudomallea), and perform bioinformatics analysis of its protein. A pair of primers was designed according to the BPSS0180 gene sequence information of B. pseudomallea K96243 strain in GenBank. BPSS0180 gene fragment was obtained by PCR amplification of B. pseudomallea hn-1 strain. The BPSS0180 gene fragment was ligated into the pET-28a(+) vector to construct the pET-28a(+)-BPSS0180 recombinant plasmid. The recombinant plasmid pET-28a(+)-BPSS0180 was transformed into E.coli DH5α competent cells, and the plasmids were identified by restriction enzyme digestion. Then, the recombinant plasmid pET-28a(+)-BPSS0180 was 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 BPSS0180 gene sequence was carried out using DNAMAN, ProtParam, SOPMA and Protscale. The results showed that the length of BPSS0180 gene was 1 146 bp; The expressed His-BPSS0180 fusion protein was about 45 ku, and was predominantly in the form of inclusion bodies; The molecular weight of the BPSS0180 protein was 40.6 ku (C₁₇₇₉H₂₈₀₉N₅₄₅O₅₃₆S₇); The extinction coefficient was 40 575; The hydrophobic index was 85.43; The instability coefficient was 46.52,which belonged to unstable protein;The theoretical isoelectric point (pI) was 5.54 and was acidic protein; The total average hydrophobicity (GRAVY) was -0.261,as hydrophilic protein; The secondary structure of the protein were mainly α-helix (58.79%) and random curl (32.02%), and its half-life of reticulocytes in mammals was predicted to be 30 h. This study provided a theoretical basis for further exploring the fuction of BPSS0180 gene of B. pseudomallei.     

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。     

Cloning,Prokaryotic Expression of BPSS1512 Gene in Goat Burkholderia pseudomallei and Bioinformatics Analysis of its Proteins

The experiment was aimed to study the clone and prokaryotic expression of BPSS1512 gene in goat Burkholderia pseudomallei and analyzed its proteins by bioinformatics. The geneome of Burkholderia pseudomallei was used as the template,and the primers were designed by DNAMAN software referring to genomic DNA sequence of Burkholoderia pseudomallei K96243 strain in GenBank (NC_006351.1).The BPSS1512 gene was amplified by PCR and the recombinant plasmid was constructed. Then the expressed protein was analyzed by SDS-PAGE and Western blotting, and the amino acid sequence encoded by BPSS1512 gene was analyzed by softwares such as DNAMAN.The results showed that the BPSS1512 gene was successfully cloned with the length of 1 425 bp,and the recombinant plasmid pET-28a-BPSS1512 was constructed. The optimum conditions for induction was that the IPTG was 10 mmol/L and 8 h for induction.The molecular weight of the protein was 53 ku,it was expressed as the form of inclusion body.In the secondary structure of BPSS15122 protein,alpha-helix,extended strand,and random coil were 24.05%,14.77% and 61.18%, respectively,and the hydrophobic core was distributed between -2.0 and +2.4 which indicated that the BPSS1512 protein was strong hydrophobicity.     

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。     

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%。     

鼠伤寒沙门菌cya基因的克隆表达及生物信息学分析

研究旨在克隆鼠伤寒沙门菌cya基因并对其进行生物信息学分析。以鼠伤寒沙门菌SL1344株为模板PCR扩增并克隆了鼠伤寒沙门菌cya基因,构建原核表达质粒pET-32a-cya,转化大肠杆菌BL21（DE3）感受态细胞,用1 mmol/L IPTG对其进行诱导表达、纯化,同时利用相关的生物信息学软件对其进行生物信息学分析。结果显示,鼠伤寒沙门菌cya基因大小为1 185 bp。经SDS-PAGE和Western blotting分析显示,在原核表达系统中主要以包涵体形式存在,蛋白分子质量大小约为58 ku。生物信息学分析表明,Cya蛋白由394个氨基酸组成,理论分子质量为44.97 ku,分子式为C₂₀₂₆H₃₁₄₆N₅₆₂O₅₇₈S₁₁,等电点（pI）为7.01,其中带负电荷残基总数（Asp+Glu）43个,带正电荷残基总数（Arg+Lys）42个。Cya蛋白无信号肽与跨膜区,为膜内蛋白,含有4个N-糖基化位点、1个O-糖基化位点、27个磷酸化位点、13个线性B细胞结合位点、11个T细胞结合位点。二级结构中α-螺旋、延伸链、β-转角、无规则卷曲所占比例分别为38.07%、19.80%、5.58%和36.55%。本试验结果为进一步研究鼠伤寒沙门菌Cya蛋白的功能及建立以Cya蛋白为抗原的免疫鉴别检测方法奠定了基础。     

多杀性巴氏杆菌中recN基因的克隆、原核表达及生物信息学分析

试验旨在对多杀性巴氏杆菌recN基因进行克隆和原核表达,并对其表达蛋白进行生物信息学分析。参照GenBank中recN基因序列(登录号:CP003313.1)设计1对引物,通过PCR扩增获得目的基因片段,构建pET-28a(+)-recN重组质粒并转化E.coli DH5α感受态细胞,提取质粒进行酶切鉴定,将鉴定正确的重组质粒转化E.coli BL21(DE3)感受态细胞,经IPTG诱导表达,对融合蛋白进行SDS-PAGE及Western blotting鉴定。结果表明,试验成功克隆了大小约为1 677 bp的recN基因序列,通过诱导表达的His-Tag融合蛋白大小约为66.94 ku,主要以包涵体形式存在。经生物信息学分析,recN蛋白的分子式为C₂₇₃₅H₄₄₂₈N₇₈₆O₈₅₅S₁₆,消光系数为24 785,不稳定系数为43.99,属于不稳定蛋白;理论等电点(pI)为5.62,为酸性蛋白;总平均亲水性为-0.316,与试验表达的包涵体蛋白性质相同,即同为疏水性蛋白;recN蛋白在哺乳动物网织红细胞的半衰期为30 h,在酵母(体内)中的半衰期>20 h,在大肠杆菌(体内)中的半衰期>10 h;二级结构主要以α-螺旋(64.87%)及无规则卷曲(21.00%)为主;经疏水性分析,预测该蛋白有3个高疏水性区域和9个高亲水性区域。本试验结果为进一步探究多杀性巴氏杆菌recN基因的功能提供了参考依据。     

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.     

中华蜜蜂GABARAP基因克隆、生物信息学分析及原核表达载体的构建

【目的】对中华蜜蜂(Apis cerana cerana)中自噬相关蛋白Atg8家族成员γ-氨基丁酸相关受体蛋白(gamma-aminobutyric acid receptor type associated protein, GABARAP)基因进行克隆鉴定和生物信息学分析,并在大肠杆菌BL21(DE3)感受态细胞中进行原核表达,以期为后续探究该基因的功能奠定基础。【方法】根据GenBank中西方蜜蜂(Apis mellifera)GABARAP基因序列(登录号:XM₀01120069.5)设计引物,采用巢式PCR对中华蜜蜂GABARAP基因进行扩增、克隆;运用生物信息学软件对其氨基酸序列相似性、二级结构、三级结构进行比对和预测分析;构建GABARAP基因原核表达载体,利用Western blotting对GABARAP蛋白进行鉴定后并用IPTG诱导纯化。【结果】经巢式PCR扩增得到中华蜜蜂GABARAP基因序列;使用在线BLAST工具对氨基酸相似性进行分析显示,中华蜜蜂GABARAP与西方蜜蜂、大蜜蜂、小蜜蜂、欧洲熊蜂、东方熊蜂的氨基酸序列相似性为100%;...     

鸭疫里默氏杆菌CAMP基因的克隆、原核表达及生物信息学分析

试验旨在克隆鸭疫里默氏杆菌协同溶血素蛋白(cooperative hemolysin protein,CAMP)基因,并对其进行原核表达和生物信息学分析。以RA贵州血清2型分离株RA-SS-8基因组为模板扩增并克隆鸭疫里默氏杆菌CAMP基因,构建pET-28a-CAMP重组原核表达质粒,转化大肠杆菌Rosetta(DE3)感受态细胞,重组菌用IPTG进行诱导表达及纯化,利用SDS-PAGE和Western blotting分析表达蛋白的特征,并运用相关生物信息学分析软件对CAMP基因进行分析。结果显示,鸭疫里默氏杆菌CAMP基因大小为1 026 bp,该基因序列与GenBank中公布的10株RA参考菌株(如RA-LZ01(CP045564.1))CAMP基因的相似性达99.7%。经BamH Ⅰ和Xho Ⅰ双酶切鉴定后,获得大小约5 369和1 026 bp的基因片段,表明pET-28a-CAMP重组表达质粒构建成功。SDS-PAGE和Western blotting分析显示,表达的重组蛋白大小约为37 ku,以可溶形式进行表达,且能与His-tag单克隆抗体在37 ku处产生特异性反应,与预期结果相符。生物信息学分析表明,CAMP蛋白分子式为C₁₆₇₀H₂₆₅₉N₄₃₇O₅₀₀S₁₂,含341个氨基酸,理论等电点(pI)为5.62,不稳定系数为40.71,表明其为不稳定的弱酸性蛋白。疏水性预测结果显示,该蛋白属于亲水性蛋白。CAMP蛋白无跨膜结构域,无信号肽,有3个O-糖基化位点,无N-糖基化位点,存在34个磷酸化位点,共有17个抗原表位。二级结构和三级结构预测结果显示,CAMP蛋白以α-螺旋和无规则卷曲为主。本试验结果为进一步研究鸭疫里默氏杆菌CAMP蛋白的功能及鸭疫里默氏杆菌病疫苗的研发提供了参考。