人源温和气单胞菌溶血素基因的克隆与表达

根据GenBank报道的人源温和气单胞菌溶血素(hly)基因序列设计引物,经PCR扩增,得到大小约为1470bp的阳性产物.利用BamHⅠ,HindⅢ位点将hly基因片段克隆到pMD19-T载体,构建得到重组质粒pWT-hly,并比较所克隆基因序列的同源性.然后将该基因克隆到原核表达载体pET-28a,构建原核重组表达质粒pWET-hly,转化BL21(DE3)并进行SDS-PAGE分析.结果表明:克隆的hly基因与GenBank报道的溶血素基因同源性为96.19%;重组菌株经IPTG诱导后,hly基因得到了高效表达.     

枯草芽孢杆菌启动子的克隆及功能验证

以pHT01穿梭质粒为骨架,构建以卡那霉素抗性基因为报告基因的启动子探针载体pHT-kan.利用该探针载体在大肠杆菌中克隆枯草芽孢杆菌168的启动子活性片段,挑取得到100个重组子.通过卡那霉素浓度梯度筛选出2个抗性最强的片段进行序列测定和分析,将启动子片段命名为BSP25、BSP31.将抗性最高的两个载体转入枯草芽孢杆菌168菌株,结果表明,它们可以在枯草芽孢杆菌中启动卡那霉素抗性基因的表达,重组菌株表现出卡那霉素抗性.     

细粒棘球绦虫重组pET30a-EgA31疫苗的构建和诱导表达

克隆细粒棘球绦虫EgA31抗原基因,构建pET30a-EgA31原核表达载体,并在大肠埃希菌宿主系统中表达EgA31重组蛋白,对表达产物进行SDS-PAGE分析.从细粒棘球绦虫成虫组织中提取总RNA,反转录生成cDNA,以此cDNA为模板RT-PCR 克隆获得EgA31抗原基因,将其克隆至pUCm-T载体,测序确定其正确性.利用定向克隆技术将EgA31抗原基因片段克隆至原核表达质粒pET30a上,转化E coli DH5α,根据选择标记的卡那霉素抗性基因筛选到阳性克隆,通过PCR分析和酶切鉴定筛选出阳性克隆.IPTG初步诱导和表达pET30a-EgA31重组蛋白,SDS-PAGE电泳检测,并经凝胶图像分析确定目的蛋白的表达水平.测序表明选取的pET30a-EgA31阳性克隆均为正确连接EgA31抗原基因的重组质粒.经IPTG诱导后重组蛋白得到成功表达,在相对分子量约为31 kDa处有表达条带,表达量约占菌体总蛋白质的26%.成功克隆并构建了pET30a-EgA31原核表达质粒.初步诱导表达出EgA31重组蛋白,为进一步研究其免疫特性奠定了基础.     

麻疯树干旱诱导差异表达基因片段的分离与功能分析

运用mRNA差异显示技术(DDRT-PCR),并结合反向Northern杂交筛选麻疯树(Jatropha.curcus)干旱胁迫下差异表达基因,最终从麻疯树叶片中分离出8条干旱胁迫诱导下表达上调的差异片段.经克隆、测序和基因信息分析后发现其中4个片段与GenBank中已知序列有较高同源性.对这4个差异表达片段的序列分析表明:JcDD-3和JcDD-13分别与1-磷酸-肌醇合成酶(MIPS)基因及甜菜碱醛脱氢酶(BADH)基因序列有较高同源性;JcDD-16与白杨中度抗旱叶片中提取的表达序列标签(CU230383)核酸序列同源;JcDD-25与植物富含亮氨酸重复序列型类受体蛋白激酶(LRR-RLK)基因序列同源.     

猪脑心肌炎病毒VR-129 B株VP2重组蛋白的构建表达及其免疫活性分析

为获得大量猪脑心肌炎VP2基因及蛋白研究的细胞模型,构建pDC315-EMCV-VP2真核表达载体,且将其转染到293T细胞,筛选出阳性质粒进行克隆。EMCV VR-129B株VP2基因序列参照GenBank(登录号:X74312),利用RT—PCR方法扩增VP2的全基因序列,将其与质粒pDC315经NheI和XhoI双酶切后连接,构建pDC315-EMCV-VP2重组表达质粒,并将其转染至293T细胞。使用荧光定量PCR方法观察pDC315-EMCV-VP2真核表达载体在细胞中的表达情况。双酶切PCR结果获得大小为780 bp的基因片段,测序结果显示与GenBank上已经公布的同名基因(登录号:X74312)序列片段同源性为100%,重组质粒构建成功。实时荧光定量PCR(QPCR)结果显示,重组质粒转染组与空质粒组之间相比,EMCV-VP2基因的表达量显著上调(P0.001);在荧光显微镜下观察转染细胞,转染成功部分出现较亮绿色荧光,EMCV-VP2基因表达稳定。从转染细胞中提取重组蛋白与EMCV阳性血清和阴性血清进行ELisa反应,具有较好免疫活性。     

羊种布鲁氏菌M5—90 omp31蛋白原核表达

克隆、测序布鲁氏菌疫苗株M5—90外膜蛋白基因OMP31,原核表达OMP31并对其检测。从羊种布鲁氏菌M5-90中PCR获得OMP31基因,连接到pBS—T克隆质粒并测序;将测序正确的基因片段克隆人大肠杆菌表达载体pET-28a,进行SDS—PAGE,而后Western—blot检测。结果M5-90疫苗株的OMP3I基因序列与羊种布鲁氏菌参考株16M的同源性为99．03％;外膜蛋白基因OMP31在大肠杆菌表达后能够被Western—blot检测到。结论：表达的布鲁氏菌疫苗株M5-90外膜蛋白OMP31可以被布鲁氏菌特异性血清识别,表现出良好的抗原性,为以后实验室诊断和疫苗的研究做好坚实的上游工作。     

中国人神经珠蛋白(NGB)基因克隆与序列分析

应用RT-PCR方法从中国人胎脑组织中扩增出神经珠蛋白(NGB)特异性编码基因片段,并将其克隆到pMD18-TVector中,构建了pMD18-T-hNGB克隆质粒,然后进行测序分析并与国外报道的NGB序列相比较.结果表明,本文获得的中国人NGB编码基因序列与国外序列的同源性为98%.     

甜味蛋白thaumatin表达载体的构建

将克隆在不同质粒上的thaumatin基因的两个片段连接起来，连接产物克隆到质粒pUC18上．测序结果表明，连接产物是一个完整的thaumatin cDNA基因．将此基因通过基因重组技术，克隆到植物表达载体pBI121中，构建了一个新的转化植物的表达载体-pBIl21-tha.     

黄粉虫抗冻蛋白基因TmAFP84的克隆和序列分析

对黄粉虫抗冻蛋白(AFPs)基因进行RT-PCR扩增后,构建克隆重组质粒pUCm-T-TmAFP84,经双酶切、PCR和测序鉴定,克隆出了与GenBank中公布的编码84个氨基酸基因(注册号AF159114)基本相一致的抗冻蛋白基因TmAFP84,其同源性高达98.81%.与其他编码84个氨基酸的抗冻蛋白基因的同源性为95.92%.进而构建了表达重组质粒pMAL-p2X-TmAFP84.     

粉尘螨Der f14基因克隆与分子特征的分析

从纯培养的粉尘螨中,根据本课题组前期合成的尘螨基因序列,将扩增的Der f14分别克隆到pUC57载体中,经扩增后用SmaI双酶切将目的片段连接到 pET-28a表达载体上得到重组质粒pET28a- Der f14.在线软件ExPaSy,NCBI网站对测序结果进行分析.分析结果表明:获得的目的基因Der f14 cDNA全长为5 013 bp.编码蛋白由1 666个氨基酸组成.粉尘螨Der f14与屋尘螨序列比对同源性达95;,信号肽序列存在1～18个氨基酸,二级结构是由a螺旋(35.83;)﹑延伸链(17.17;)﹑无规则区(47;)组成.目的蛋白是一种卵黄蛋白原.     

黄孢原毛平革菌锰过氧化物酶基因（MnP2）的克隆

应用RT-PCR技术,从黄孢原毛平革菌5.766（Phanerochaete chrysosporium）总RNA中成功扩增出预期大小约为1.3 kb的特异性条带,将扩增产物提纯后克隆入PUC19载体,经转化、筛选及酶切鉴定后,获得锰过氧化物酶（MnP2）基因的克隆。序列分析表明,扩增的MnP2基因片段其cDNA长度为1 149 bp,编码358个氨基酸,与其它已发表文献报道的MnP2序列一致,与其同工酶MnP1和MnP3的核苷酸同一性分别为81%和66%。     

pQE30-bgln原核表达质粒的构建和鉴定

构建重组原核表达质粒pQE30-bgln使之可以在大肠杆菌中表达以获得β-葡糖苷酶,用于提高从植物中提取白藜芦醇的得率.以克隆质粒pUCP67为模板,用降落PCR的方法扩增β-葡糖苷酶基因片段(bgln).利用原核表达载体pQE30构建pQE30-bgln重组质粒,用酶切电泳验证重组结果的正确性,测序检测质粒重组后序列情况.PCR结果显示扩增片段2.2 kb,与预期相同.重组质粒酶切后显示其大小约5.6kb,大小及酶切图谱与预期相同.经测序发现插入片段读码框正确.可用于原核表达的pQE30-bgln质粒构建成功.     

SpltNPVp49基因的克隆和序列分析

根据新发现的杆状病毒凋亡抑制基因ｐ４９基因的序列设计了一对引物,以ＳｐｌｔＮＰＶ基因组ＤＮＡ为模板,通过ＰＣＲ扩增获得了预期大小的约１．３ｋｂ的ＤＮＡ片段,将此片段克隆到ｐＧＥＭ－Ｔ载体上并直接测序。序列分析表明,该片段为ＳｐｌｔＮＰＶ完整的ｐ４９基因开放读码框。与已知的杆状病毒ｐ４９基因的序列同源性为８７％,与之对应的氨基酸序列的同源性高达９３％。它是首次从ＳｐｌｔＮＰＶ中克隆到的抑制细胞凋亡的     

新疆绵羊种布鲁氏菌GroEL基因的克隆及序列分析

参考牛种布鲁氏菌的GroEL（热休克蛋白）基因设计引物,扩增出新疆绵羊种布鲁氏菌GroEL基因片段,将其片段克隆到T载体上测序。结果表明：新疆源布鲁氏菌GroEL基因片段长1641bp,编码546个氨基酸,与羊种（B．melitensis）、猪种（B．suis）以及牛种（B．abortus）GroEL基因的核苷酸序列同源性分别为99．88％、99．82％、99．88％,推导的氨基酸序列同源性在99％以上,显示了很强的保守性。     

PRV广东株gE基因去信号肽片段的克隆与序列测定

根据伪狂犬病病毒（PRV）Rice株gE基因的序列设计并合成了1对引物,以我国PRV地方毒株广东株的基因组DNA为模板,通过PCR方法获得了一大小约1.6kb的DNA片段,并将其克隆到pMD18-T载体上进行测序,序列测定结果显示,该片段长1665bp,编码555个氨基酸,与PRV Rice株gE基因的核苷酸序列同源性为97.7%,氨基酸序列同源性为95.9%。     

中度嗜盐菌GbsB基因的克隆、序列分析及生物信息学分析

为构建耐盐的转基因植物提供材料,根据地衣芽孢杆菌Bacillus licheniformisATCC 14580的GbsB基因的核甘酸序列设计1对特异性引物,通过PCR的方法扩增由分离的中度嗜盐菌Bacillus sp.XJ1-05的GbsB基因,经T/A克隆,插入到pBS-T载体上进行序列测序,采用NCBI-BlastX软件在Genbank数据库中进行同源性检索,结果表明:得到的GbsB基因的开放阅读框(ORF)全长为1209bp,编码1个由402个氨基酸残基组成的蛋白质;其与Bacillus licheni-formisATCC 14580的GbsB基因的氨基酸序列同源性高达96%。生物信息学分析表明:该GbsB基因编码蛋白无信号肽,无跨膜区域,高亲水性,有2个乙醇脱氢酶作用位点。     

人类Neuritin cDNA的克隆和表达

从人胎脑cDNA文库筛选出一条1618bp的cDNA。此cDNA含有一个426bp的最大开放阅读框,编码一个142个氨基酸的蛋白质,预测分子质量为15.3ku。与目前数据库中序列比较,该cDNA与鼠neuritin基因同源性达98％。多组织Northern blot分析显示neuritin在脑组织高度表达。neuritin cDNA的读框片段正确插入到pQE40表达载体中,获得了预期的表达产物,并初步得到了其纯化蛋白。     

Expression of the intact C<Subscript>4</Subscript> type<Emphasis Type="Italic">pepc</Emphasis> gene cloned from maize in transgenic winter wheat

Maize intact C4-pepc gene was amplified through LA-PCR and successfully sub-cloned into modified vector pGreen0029 to form a stable expression construct named as pBAC214 (12 kb), which contains CaMV 35S promoter driven bar gene as selection marker. Comparing the cloned DNA sequences (6.7 kb) with published maize C4-pepc gene (GenBank accession E17154) sequences, the identity of DNA sequence alignment is 98.96%. There are only 49 differences between these two intact DNA sequences, of which 13 occur in the region of promoter, 18 in introns, and 18 in exons. The homology of mRNA sequence alignment is 99.38%, and the putative amino acids sequence identity is 99.38%. There are only 15 differences between these two mRNA, and these differences bring 4 sites mutant on the putative amino acids of PEPC protein. Through biolistic bombardment of PDS1000/He system, expression vector pBAC214 has been transformed into winter wheat. Southern blotting results show that the intact C4-pepc gene has been integrated into genome of winter wheat. SDS-PAGE analysis of leaf soluble protein in transgenic wheat showed that the intact C4opepc gene was well transcribed, spliced and translated as in maize. The enzyme activity of leaf PEPC in transgenic wheat has been detected. The activities of leaf PEPC increased over 3-5 times in some transgenic plants. The data of photosynthesis rate and transpiration rate of transgenic wheat flag leaves showed that the C4-pepc gene can increase the photosynthesis rate and transpiration rate of transgenic wheat.     

Cloning and characterization of vernalization-related gene (ver203F) cDNA 3' end

Based on the cDNA fragment sequence of vernalization-related gene verc203 cloned by differential screening in our lab, the 5' primer has been designed. The cDNA 3' end of ver203 gene (1 197 bp) has been cloned by the RACE method. And it is identified by Northern blotting that its expression is special in vernalization treatment. After comparing the sequence in the nucleotide sequence databases of Genbank, EMBL and DDBJ, the gene has homology with Hordeum vulgare jesmonate-induced protein gene. It is suggested that this gene might be related to the signal transduction mediated by jamonate.     

cDNA cloning and expression of earthworm fibrinolytic enzyme component A

Earthworm fibrinolytic enzyme component A(EFEa),a protein with dual fibrinolytic activity ,is one of the major therapeutically important earthworm fibrinoltic enzyme components .The cDNA fragment encoded the mature protein was cloned from earthworm (Eisenia fetida )by the RT-PCR technique,The deduced amino acid sequence of the EFE component A show high homology with some members of serine proteases trypsin family,and the amino acid residues constituting the active sites are conserved in the EFEa as compared with the other proteins of the trypsin family ,The cDNA fragment was subcloned into the expression vector pQE31 and pMAL-c2X of E.coli.The resulting expression plasmids,pQE-efea and pMAL-efea ,were used to transform the E.coli strain M15.Recombinant protein bands corresponding with calcuated molecular witht were induced .The induced His6-EFEa fusion protein with pQE-efea was accumulated into inclusion body ,while the induced MBP-EFEa fusion protein with pMAL-efea was soluble and showed fibrinoloytic activities.