金黄色葡萄球菌FnBP配体结合区基因的克隆及其原核表达

[目的]克隆金黄色葡萄球菌FnBP配体结合区基因,并构建原核表达载体,进行原核表达：方法]设计引物,采用PCR方法扩增FnBP配体结合区基因,BA克隆后,构建了克隆质粒pMD18-FnBP。用BarnHI和EcoRI双酶切pMD18-FnBP和pET28a（＋）,将纯化的基因FnBP亚克隆至pET28a（＋）,构建重组表达质粒pET28a-FnBP,并将其转化至大肠杆茼感受态B121（DF3）中,IPTG诱导表达,并对表达产物进行分析。[结果]PCR扩增出1条约370hp的目的片段,表达产物经SDS-PAGE分析,在30kDa处出现了与目的蛋白一致的外源蛋白带．Westem blot分析表明该蛋白具有金黄色葡萄球菌的抗原性。[结论]已成功构建了FnBP配体结合区基因,并在原核细胞中表达。     

黑白花奶牛白细胞介素-2基因的克隆和表达(英文)

[Objective] The aim of this study is to clone bovine interleukin-2 gene(IL-2)and observe its expression in prokaryotic cells.[Method]Bovine IL-2 gene was amplified from total RNA of peripheral blood lymphocytes of holstein-friesian cows by RT-PCR.Subsequently,the gene was cloned into pGEX-2T prokaryotic expression plasmid to construct recombinant,which was then transformed into Escherichia Coli BL21.After IPTG induction,SDS-PAGE analysis was conducted.[Result]A 500 bp target fragment corresponding with expectation was obtained by RT-PCR.The cloned gene successfully expressed fusion protein of about 43 kD in prokaryotic cells.[Conclusion]This study provided a theoretical and material basis for further researches on IL-2 gene.     

铜绿假单胞菌脂肪酶Lipase基因的原核表达(英文)

[Objective] The aim of this study was to investigate the prokaryotic expression of pseudomonas aeruginosa Lipase gene.[Method]Lipase gene was amplified by PCR from the genome DNA of pseudomonas aeruginosa,and its nucleotide sequence was determined.The prokaryotic expression vector of Lipase gene was constructed by the gene recombination technique.The protein expression was induced for 4 hours by IPTG with the final concentration of 1.0 mmol/L,and then SDS-PAGE electrophoresis was analyzed.[Result]The sequence of mature peptides in Lipase gene cloned from pseudomonas aeruginosa had a 99.36% homology with that of pseudomonas aeruginosa lipase submitted in NCBI,so the prokaryotic expression vector of Lipase gene pET32a-Lip was successfully constructed.Furthermore,the results of SDS-PAGE electrophoresis showed that the target gene was expressed highly and effectively.[Conclusion]The cloned pseudomonas aeruginosa lipase with its signal peptide could be normally expressed in E.coli and also used for further study.     

猪细小病毒(PPV)SD1株NS1基因的克隆与原核表达

为研究猪细小病毒（Porcine Parvovirus，PPV）NS1基因，建立快速、准确诊断猪细小病毒的方法，减少猪细小病毒造成的损失，笔者克隆了含有NS1基因的片段，并且与已登录猪细小病毒的相应序列进行比较，成功构建了原核表达重组质粒PET 30a／NS1，并在E.coli中进行了诱导表达。     

金黄色葡萄球菌的群体效应研究进展

金黄色葡萄球菌(Staphylococcus aureus)中已知的群体效应系统是Agr系统和LuxS/AI-2系统,其在细菌浓度等于或大于1×10~7 CFU/mL时会被激活,通过调控相应基因,间接或直接调控生物被膜的产生和降解、细菌毒素的分泌以及细菌的生长。综述了金黄色葡萄球菌的群体效应研究进展,旨在为今后对金黄色葡萄球菌的研究寻找新方向,金黄色葡萄球菌致病性及生物被膜的形成与金黄色葡萄球菌的群体效应系统之间存在必然联系。     

牛瑟氏泰勒虫P23基因的克隆与生物信息学分析(英文)

[Objective] The aim of this study is to provide basis for developing genetic engineering vaccine and diagnostic kit for Theileria sergenti infection. [Objective] P23 gene of Theileria sergenti was amplified from its genomic DNA by PCR amplification, and cloned into the pGEM-Easy vector; then the sequencing result was analyzed with bioinformatics methods. [Result] Whole length of the P23 gene from Theileria sergenti is 684 bp containing a 672 bp open reading frame. The deduced amino acid sequence (223 amino acid residues) contains a signal peptide of 19 amino acid residues and two fragments of transmembrane domains, with relative molecular weight of the 25.886 kD and with the pI of 9.22. The homology between the yielded sequence and Chitose of Theileria sergenti P23 gene(TS-Chitose type, D84446), Ikeda of Theileria sergenti P23 gene(TS-Ikeda type, D84447) reached 99% and 90%, respectively. The sequence has been accessed in GenBank(EU573168). [Conclusion] The protein encoded by the P23 gene has better stability and immunogenicity, thus can be used as the antigen candidate for preparing genetic engineering vaccine for Theileria sergenti.     

棉铃虫蛋白酶体β5基因Habeta5的克隆与序列分析(英文)

[Objective] Using molecular biotechnology to clone the proteasome β5 gene from cotton bollworm (Helicoverpa armigera), this research aimed to provide basis for further research on the function of proteasome β5 gene in cotton bollworm. [Method] Total RNA was extracted from midgut of cotton bollworm. The full length cDNA of Habeta5 gene was cloned by using rapid amplification of cDNA ends (RACE) technology, then sequence analysis was carried out. [Result] The full length cDNA sequence was successfully cloned and isolated, named as Habeta5. It was 947 bp in length, contained an ORF (843 bp) and encoded 280 amino acid residues, with the predicted mass of 30.87 kD and isoelectric point(pI) of 9.60. In the deduced amino acid sequence, a proteasome β5 subunit domain lies between 74th to 261st amino acid residues. It has more than 62% identity to other insects such as Drosophila melanogaster. The proteasome β5 subunit conservative regions were very similar with each other. Molecular evolution by Neighbor Joining method indicated that Habeta5 was homologous with other proteasome β5 subunit of species. [Conclusion] Sequence alignment shows that the cloned fragment is a proteasome β5 subunit gene (GenBank accession number: FJ358434).     

果汁饮料加工过程中金黄色葡萄球菌生物被膜形成的研究

从清洗过的果汁饮料加工设备的管件中采样分离细菌,对纯化后的菌株进行了形态和生化反应鉴定以及分子生物学鉴定;同时分别采用银染法和结晶紫染色法对分离菌株所形成的生物被膜进行了检测.结果表明,分离菌株被鉴定为金黄色葡萄球菌(Staphylococcus aureus);银染法和结晶学染色法均证明金色葡萄球菌能形成生物被膜,从而证明清洗后的果汁饮料加工管件中依然可能形成金色葡萄球菌生物被膜.     

金黄色葡萄球菌多药耐药基因norA的克隆及其原核表达

按金黄色葡萄球菌多重耐药转运蛋白NorA的编码序列设计引物，以金黄色葡萄球菌基因组DNA为模板，扩增出norA基因中1155bp cDNA片段，将所得片段与pMD18-T载体连接，转化到感受态大肠杆菌JM109中，成功地筛选到阳性克隆，其质粒测序结果与文献报道一致。从阳性克隆中提取质粒，经Nde Ⅰ和XhoⅠ酶切，回收1155bp目的片段，定向克隆到pET28a（＋）表达载体中，转化感受态大肠杆菌DH5a，提取质粒，再次转化到BL21（DE3）中，成功地筛选到阳性克隆。经IPTG诱导阳性菌，通过SDS-PAGE检测出norA基因的表达。     

O型口蹄疫病毒结构蛋白基因VP1的克隆与原核表达(英文)

According to the complete genome of foot-and-mouth disease virus(FMDV)type O,a pair of special primers was designed to amplify VP1 gene.The VP1 gene was amplified by RT-PCR and subsequently inserted into the expression vector pGEX-6p-1 and induced by IPTG.Then SDS-PAGE showed the expressed protein was 51 kD in molecular weight.Then the product was purified by GSTrap FF columns.The product was detected through Western-blot that showed the protein has antigenicity.It provided fundamental data and materials for further investigation on diagnosis method of FMDV.     

金黄色葡萄球菌wood46株eno基因的克隆及原核表达

为获得Eno的重组蛋白,采用PCR法从金黄色葡萄球菌wood46株基因组扩增eno基因,并连接到pMD18-T载体上,转化至BL21感受态细胞中;重组质粒经PCR及酶切鉴定后,送上海生工测序。将鉴定正确的质粒酶切回收产物与原核表达载体pET-32a连接,然后转化至BL21感受态细胞,对平板筛选的阳性质粒进行PCR和酶切鉴定。对鉴定正确的重组菌进行诱导表达并纯化。实验结果表明成功构建了pET32a-eno表达载体,并获得了该纯化蛋白。该实验结果为S.aureus亚单位疫苗研究奠定了一定的基础。     

油菜COR基因的克隆及原核表达

以冬油菜陇油6号叶片为材料,利用RT-PCR法克隆到油菜COR基因编码区序列,全长390 bp。将其与大肠杆菌表达载体p ET-30a连接,构建原核表达载体p ET-30a-COR,并转化大肠杆菌BL21,经IPTG诱导表达后,SDS-PAGE检测结果表明该基因表达了1个约14.3 k D的蛋白,为进一步研究目的蛋白的结构和功能提供了实验基础。     

犬新孢子虫NcSRS2基因片段的克隆及原核表达

以含有犬新孢子虫NcSRS2基因的质粒pMD18-NcSRS2为模板,应用PCR方法扩增Nc-SRS2基因,将该基因片段克隆至原核表达载体pGEX-4T-2,构建了重组表达质粒pGEX-Nc-SRS2,转化大肠杆菌BL21中并诱导表达。结果显示,克隆的基因片段长1100bp,SDS-PAGE、Western blotting分析显示,重组质粒pGEX-NcSRS2在大肠杆菌中得到了高效表达。     

猪圆环病毒2型ORF2基因片段的克隆与原核表达

为在原核表达系统中表达猪圆环病毒2型(PCV2)ORF2基因片段,提取感染PCV2的细胞基因组DNA,PCR扩增ORF2基因片段并克隆至pMD-18T载体上,经PCR、酶切及测序鉴定后,将该基因重组至pET-28a原核表达载体,构建了表达载体pET-28a-ORF2。该重组质粒在大肠杆菌BL21中经1mmol/LIPTG于37℃诱导5h得到最佳表达。表达重组蛋白的分子量为26.0kD,以包涵体形式存在。Western-Blot分析表明,该重组蛋白与PCV2阳性血清发生特异性反应,表明该重组蛋白具有良好的反应原性。     

猪细小病毒自然弱毒N株VP2基因的克隆及原核表达

为了进一步研究猪细小病毒自然弱毒株(PPV-N株)的自然弱毒分子生物学机理,对PPV-N株VP2基因进行克隆、测序和原核表达研究.结果表明,成功构建PPV-N株VP2基因的克隆重组质粒pMD18-T-VP2及表达重组质粒pET32a-VP2,经SDSPAGE及Western blotting鉴定,PPV-N株VP2基因在BL21(DE3)plysS菌中成功进行融合表达,表达出约85.4 KDa的VP2融合蛋白,且表达的VP2融合蛋白能与PPV阳性血清发生特异性反应.PPV-N株VP2基因的成功克隆及原核表达为今后研究PPV-N株的自然弱毒的分子生物学机理和研制PPV诊断抗原奠定了基础.     

猪细小病毒(PPV)SD1株NS1基因的克隆与原核表达

[目的]为建立猪细小病毒的快速诊断方法提供理论依据。[方法]根据GenBank上猪细小病毒基因组序列和原核表达质粒pET30a(+)多克隆位点序列设计1对引物,应用PCR技术扩增出猪细小病毒SD1株NS1基因全序列,对阳性重组质粒进行测序和同源性比较。构建原核表达重组质粒pET 30a/NS1,并在大肠杆菌中进行诱导表达。[结果]通过PCR扩增获得2 208 bp目的片段。所克隆NS1基因与已报道的PPV相应基因的核苷酸同源性为97.3%~99.4%,表明NS1基因具有高度保守性,但在偏3′端连续缺失12个碱基。所克隆的PPVNS1基因在原核细胞中成功表达,其表达产物主要以包涵体形式存在。[结论]SDS-PAGE检测结果表明PPVNS1蛋白的分子量为86 kD。     

水牛朊蛋白成熟片段基因的克隆与原核表达

【目的】克隆、分析水牛朊蛋白（prion protein，PrP）成熟片段基因，并获取纯化的表达产物。【方法】将应用PCR技术扩增的水牛成熟PrP的核酸序列克隆到表达载体pET-32a，并分析其序列；把阳性克隆质粒pET-32a-BPrP转化表达菌BL21(DE3)，鉴定纯化的表达产物。【结果】水牛朊蛋白成熟片段核酸序列及其推导的氨基酸序列与已知其他牛科动物的成熟PrP核酸、氨基酸序列间的相似性分别在97.1%和97.2%以上，并且发现水牛成熟PrP有5个重复序列，编码的氨基酸序列其中有2个九肽和3个八肽重复序列。具有较高的表达水平的PrP融合蛋白被纯化后，用Western印迹实验证明该蛋白具有PrP抗原活性。【结论】首次报道了克隆、分析水牛朊蛋白成熟片段基因，发现水牛成熟PrP有5个重复序列，并获得了具PrP抗原活性的水牛成熟PrP融合蛋白。