四川白鹅白细胞介素-2基因的克隆及生物信息学分析(英文)

[Objective] The study aimed to clone interleukin-2(IL-2) gene from Sichuan white goose. [Method] Based on the IL-2 gene of duck accessed in GenBank, a pair of primers was designed for cloning IL-2 gene from total RNA of peripheral blood lymphocytes of Sichuan white goose stimulated by ConA via RT-PCR technology. The yielded fragment was sequenced for bioinformatics analysis. [Result] The full length of IL-2 gene of Sichuan white goose is 468 bp that contains a 441 bp open reading frame(ORF), encoding 146 amino acid residues. Bioinformatics analysis shows that the amino acid sequence of IL-2 gene of Sichuan white goose contains four phosphorylation sites, a glycosylation site and a signal peptide with 21 amino acid residues. Homologies of IL-2 nucleotide sequence and amino acid sequence between Sichuan white goose and duck, chicken, turkey are 92.7%, 77.5%, 78.2% and 85.8%, 65.5%, 64.1%, respectively. By contrast IL-2 nucleotide sequence and amino acid sequence between Sichuan white goose and mammalian and rodents such as human, monkey, rat, bovine, horse, pig, cat, mouse, rabbit and deer, are all less than 45% and 28%, respectively. [Conclusion] The IL-2 gene of Sichuan white goose has closer genetic relationship with those of chicken and duck.     

黑白花奶牛白细胞介素-2基因的克隆和表达

[目的]克隆牛白细胞介素-2基因(IL-2),并观察其在原核细胞中的表达。[方法]应用RT-PCR技术从黑白花奶牛外周血淋巴细胞总RNA中扩增编码牛IL-2基因,并将其克隆到pGEX-2T原核表达质粒中,转化大肠杆菌BL21,经IPTG诱导后进行SDS-PAGE分析。[结果]通过RT-PCR扩增获得500 bp的目的片段,所克隆的IL-2基因在原核细胞中成功表达,表达产物为分子量约43 kD的融合蛋白。[结论]该研究为IL-2基因的进一步研究提供了理论依据和物质基础。     

金黄色葡萄球菌FnBP配体结合区基因的克隆及其原核表达(英文)

[Objective] The study aimed to clone the FnBP ligand binding gene of Staphylococcus aureus and run prokaryotic expression by constructing a prokaryotic expression vector. [Method] The gene encoding FnBP ligand binding gene was amplified from S.aureus chromosomal DNA by PCR technique. After T-A cloning, plasmid pMD18- FnBP was constructed. pMD18- FnBP and pET28a(+)were digested by BamH Ⅰ and EcoR Ⅰ double enzymes, then the purified FnBP ligand binding gene was subcloned into the expression vector pET28a(+), and the prokaryotic expression vector pET28a-FnBP was thus constructed. The constructed plasmid pET28a-FnBP was transformed into Escherichia coli BL21(DE3) competent cells. The bacterium was induced by IPTG and the expressed products were analyzed by SDS-PAGE and Western blot. [Result] The gene fragment with the length of 370 bp was amplified by PCR approach. One approximately 30 kD exogenous protein was observed in SDS-PAGE analysis. Western blot analysis indicates the protein has antigenicity of S.aureus. [Conclusion] The FnBP ligand binding gene of S.aureus was successfully cloned and expressed in prokaryotic cells.     

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.     

猪白细胞介素-2基因的克隆与表达

根据GenBank上发表的猪白细胞介素-2（Interleukin．2,IL-2）基因序列,设计1对引物。采用RT-PCR技术,以ConA刺激的猪外周血淋巴细胞为材料,从总RNA中扩增出484bp的特异性片段,将其克隆入pGEM-Teasy载体。序列测定表明;扩增片段为猪IL-2基因,该基因与GenBank上发表的猪IL-2基因的核苷酸序列同源性达99．8％。将其定向克隆至原核表达载体pET32a,构建了表达重组猪IL-2的基因工程菌株,经IPTG诱导表达和层析纯化,获得了纯化的重组猪IL-2蛋白。     

奶牛白细胞介素-2基因的克隆及序列分析

根据GenBank上发表的牛IL-2(M13204)基因序列,应用Primer Premier5引物设计软件自行设计一对引物,以ConA刺激培养24 h的奶牛外周血淋巴细胞为材料,从总RNA中扩增出奶牛IL-2基因,然后测序.结果表明克隆的奶牛IL-2基因大小477 bp,编码158个氨基酸,与GenBank上发表的牛IL-2基因序列比较,其核苷酸的同源性99.4%,与马、猪、猫、狗、山羊、绵羊的核苷酸的同源性分别为31.8%,32.0%,31.6%,30.3%,30.8%,31.0%.     

长毛兔和獭兔白细胞介素-2基因的克隆与真核表达

采用RT-PCR法从ConA诱导培养的德系安哥拉长毛兔和白色獭兔外周血淋巴细胞总RNA中扩增得到白细胞介素-2基因(IL-2),经序列测定表明,长毛兔和獭兔IL-2基因大小均为462 bp,两序列碱基组成无差异,该基因编码一个由153个氨基酸组成的多肽,包括20个氨基酸残基的信号肽和133个氨基酸残基的成熟肽。德系安哥拉长毛兔和白色獭兔IL-2基因间同源性为100%,与欧洲野兔及中国白兔的IL-2基因同源性分别为99.4%和98.9%。与GenBank中报道的猿、人、猕猴、狒狒、猫、马、大熊猫、犬、猪、水牛、山羊和鼠的IL-2核苷酸同源性分别为86.9%、86.7%、86.2%、86.1%、84.3%、84.3%、82.8%、82.6%、81.4%、76.9%、76.7%、75.0%。进化树分析发现,兔和猿的IL-2亲缘关系最近。通过脂质体法转染COS-7细胞,获得具有一定生物学活性的IL-2蛋白。     

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。     

鸡白细胞介素-2基因的克隆与酵母表达质粒的构建

从经ConA活化的4周龄岭南黄鸡脾脏淋巴细胞中分离提取总RNA,经过反转录PCR(RT-PCR)扩增出鸡白细胞介素-2(1L-2)cDNA片断,PCR产物克隆至pGEM-TEasy载体,重组质粒命名为IL-2-T,经菌落PCR与限制酶切鉴定,然后测序,结果表明克隆片断长度为432bp,与预期大小一致,为鸡IL-2基因的编码区全长,将IL-2-T克隆重组质粒进行双酶切(Cla I与Xba I),回收IL-2片断插入同样酶切的毕赤酵母表达质粒pPiCZa-C,构建重组IL-2-C表达质粒,为鸡IL-2在毕赤酵母中的表达奠定基础。     

黄牛白细胞介素-2基因的克隆与序列分析

为了克隆黄牛IL-2基因,采用RT-PCR技术,用ConA刺激12 h的黄牛颈部外周血淋巴细胞为材料,从总RNA中扩增出黄牛IL-2基因,琼脂糖凝胶电泳技术显示扩增片段约为500 bp,分离纯化,将其克隆到PDM18-T载体,重组质粒经PCR鉴定、双酶切鉴定以及核苷酸测序。结果显示,克隆的黄牛IL-2基因大小468 bp,与GenBank中报道的水牛、牛、绵羊、猫、马、鸡、犬、人、山羊、鼠、猪IL-2基因比较,同源性分别为98.9%;98.5%;97.0%;82.4%;79.9%;5.3%;78.2%;79.7%;95.9%;65.2%;84.7%。     

犬IL-2基因克隆及原核表达

[目的]研究犬IL-2基因的克隆和表达,为开发新型免疫增强剂和基因工程疫苗提供理论支持。[方法]从犬全血中分离出白细胞,经刀豆素刺激培养20 h后,提取总RNA;根据GenBank中犬IL-2基因序列,设计1对引物,进行PCR扩增,并构建原核表达载体pET-28a,将其转化到宿主菌BL21中,经IPTG诱导表达,表达产物用SDS-PAGE进行分析。[结果]RT-PCR扩增出一条约500 bp的条带,与预期结果相符;重组质粒pMD18-T-IL2经双酶切后,产生一个约500 bp基因片段,说明目的基因被成功克隆;表达载体pET-28a-IL2经双酶切和PCR鉴定后,分别产生一个约500 bp目的片段,表明表达载体构建成功;表达产物经SDS-PAGE分析,分子量约为20 kDa,与预期蛋白大小基本相同。[结论]犬IL-2基因被成功克隆和表达。     

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。     

陕北白绒山羊Lhx2基因cDNA序列的克隆与原核表达

根据GenBank中已发表的Lhx2基因序列设计引物,采用RT-PCR方法,从陕北白绒山羊皮肤组织克隆Lhx2基因编码区cDNA,对其进行生物信息学分析,并构建原核表达载体pET-Lhx2,在大肠杆菌BL21(DE3)中表达。最终获得陕北白绒山羊Lhx2基因,长1 221bp;酶切和测序结果显示,原核表达载体pET-Lhx2构建成功;SDS-PAGE和Western blot分析表明,重组原核表达载体在大肠杆菌中成功表达出目的融合蛋白。     

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.     

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。     

蜡梅花香基因SAMT的cDNA克隆及在大肠杆菌中的表达(英文)

[目的]研究蜡梅香基因SAMT的cDNA克隆及在大肠杆菌中的表达。[方法]以蜡梅花瓣总RNA为模板进行RT-PCR,扩增得到与预期大小相同的PCR产物带,回收RT-PCR产物,将其与PMD18-T载体进行T-A克隆连接并导入大肠杆菌DH5,经菌落PCR和双酶切鉴定,筛选带有目的基因的重组质粒并进行测序。[结果]测序证实成功克隆得到蜡梅花香基因SAMTcDNA的ORF片段,其长度为1196bp,编码380个氨基酸残基,与已报导的蜡梅SAMT(ABU88887)的同源性为99.2%将SAMT基因亚克隆到原核表达载体PGEX4T-1中,转化大肠杆菌DH5α获得其原核表达菌株pGSAMT;采用0.01mol/L的IPTG进行诱导表达,经SDS-PAGE分析,SAMT的融合表达蛋白分子量约为66kDa,与预期的26KDa的GST带和42.3KDa的蜡梅SAMT基因编码蛋白构成的融合蛋白大小接近。[结论]该研究成功克隆并表达了蜡梅花香基因SAMT。     

猪源ETEC的黏附素基因克隆与原核表达

[目的]克服传统方法制备黏附素抗原的缺点。[方法]应用PCR对猪源性产肠毒素性大肠杆菌(ETEC)的黏附素F4、F5和F6的质粒中扩增出faeG、fanC和fasG基因片段,克隆测序并与pET 32a(+)构建重组表达载体。将重组表达载体转化E.coli表达菌株BL21(DE3),并分析其免疫原性。[结果]重组表达载体经IPTG诱导获得高效表达。血凝抑制试验表明,鼠抗血清能抑制标准的ETEC强毒株凝集红细胞,抑制效价在1∶128以上。这说明克隆表达的猪源ETEC黏附素蛋白具有良好的免疫原性。[结论]该研究可为进一步开发抗体制剂奠定基础。     

牛结核杆菌mpb-83基因的扩增和表达

采用PCR方法扩增牛结核杆菌mpb-83基因,并将其连接到T3克隆载体,然后克隆到原核表达载体PET-32a中,构建重组质粒PET-32a—mpb-83。以重组质粒转化大肠杆菌BL21,用NI柱纯化,最后纯化产物在SDS—PAGE中检测。结果得到约664bp的目的片段,且MPB-83可在大肠杆菌BL21中高效表达。