禽白血病病毒p19基因末端片段在大肠杆菌中的表达

根据禽白血病病毒（ALV）p19基因末端序列合成一条82 bp的双链DNA片段,将其克隆到表达质粒pGEMEX-1中,序列分析结果与设计的相符。重组表达质粒转化的大肠杆菌BL21（DE3）经IPTG诱导后产生34kD融合表达产物,与理论值相符;Western-blot分析表明该表达产物能与兔抗ALV血清发生反应。     

A亚群禽白血病病毒囊膜基因gp85片段在大肠杆菌中的表达

将RAV－1囊膜基因gp85片段来克隆到表达质粒pET-21d( )中得到重组表达质粒pET-21d-RAV-1env(BelⅡ/SalⅠ）序列分析表明该插入片段的核苷酸序列和阅读框都与RAV－1囊膜基因相应序列相同。用其转化大肠杆菌BL21（DE）3并经IPTG诱导,SDS－PAGE分析表明RAV－1囊膜基因融合蛋白表达产物约20kD,与理论值相符;IPTG诱导起始时间比诱持续时间对表达量的影响更大。     

A亚群禽白血病病毒囊膜基因gp85片段在大肠杆菌中的表达

将RAV-1囊膜基因gp85片段亚克隆到表达质粒pET-21d(+)中得到重组表达质粒pET-21d-RAV-1env(BglII/SalI),序列分析表明该插入片段的核苷酸序列和阅读框都与RAV-1囊膜基因相应序列相同.用其转化大肠杆菌BL21(DE3)并经IPTG诱导,SDS-PAGE分析表明RAV-1囊膜基因融合蛋白表达产物约20kD,与理论值相符;IPTG诱导起始时间比诱导持续时间对表达量的影响更大.     

球毛壳菌（Chaetomium globosum）过氧化物膜蛋白过敏原基因克隆、序列分析及原核表达

以球毛壳菌cDNA文库中获得过氧化物膜蛋白（pero）基因片段（GenBank Accn:BP099709）为基础,用RACE 技术获得该基因的全长cDNA序列。序列长747bp,由412bp的3′RACE产物和508bp的5′RACE产物拼接而成。开放阅读框501bp,编码166个氨基酸,蛋白分子量为17.5kD,理论等电点为5.75。利用cDNA两侧非编码区序列作引物克隆出该基因的DNA序列,序列分析表明该基因由2个内含子和3个外显子组成。ClustalX多序列比对表明：该基因与粗糙脉孢菌（Neurospora crassa）的过氧化物膜蛋白过敏原同源性最高（83%）。将pero基因编码区克隆到原核表达载体pET28a中,构建成表达质粒pET28a-pero并转化大肠杆菌BL21,IPTG诱导后SDS-PAGE检测表达情况,结果发现在21kD处有一特异性融合蛋白带,大小与预期相符,说明该基因已经在大肠杆菌中表达。克隆的cDNA序列、DNA序列及推测的氨基酸序列在GenBank登录(登录号分别为AY555771,AY584753,AAS66898)。     

黑曲霉W3350脯氨酰内肽酶在大肠杆菌中的表达

目的：构建pET-PEP重组原核表达载体,并对表达产物进行鉴定。方法：采用RT-PCR技术从黑曲霉（Aspergillus niger）W3350中扩增出脯氨酰内肽酶（PEP）的基因,插入表达载体pET-22b（＋）,经EcoRⅠ和HindⅢ双酶切和DNA序列测定验证,将正确的重组质粒转入大肠杆菌BL21（DE3）中,IPTG（终浓度1mmol/L）诱导获得表达。结果：表达产物进行超声破碎,经SDS-PAGE电泳检测,PEP分子质量大约为57kDa,与理论值相符,通过酶活方法测定脯氨酰内肽酶酶活为0.656U/ml,约是出发菌株的5倍。结论：首次成功构建表达载体pET-PEP并在原核细胞中表达,为进一步研究PEP的生物学功能奠定了基础。     

刚地弓形虫RH株果糖1，6-二磷酸醛缩酶的基因序列分析与融合表达

目的：对编码刚地弓形虫（Toxoplasma gondii）RH株果糖1,6-二磷酸醛缩酶基因进行克隆和序列分析,并构建原核栽体pETcFN融合表达该重组蛋白,以研究该分子在弓形虫速殖子入侵中的作用。方法：从刚地弓形虫RH株速殖子cDNA中扩增编码果糖1,6-二磷酸醛缩酶的基因,并克隆到T载体上,测序确认;通过PCGENE及在线工具对此编码序列进行分析;将该基因亚克隆到表达栽体pET30a（＋）上,构建质粒pETcFN;表达产物用Western blot进行进一步确认。结果：经PCR鉴定和质粒双酶切鉴定后测序,确认插入T栽体的序列为所需的目的序列。根据序列分析,推断蛋白的预计分子量为39．2kDa,等电点为7．75;BLAST分析,发现同源性52％以上的序列32条;模序分析发现其分别在223～233、21～147及15～363位置隐含有与PS00158（PmfileScan）、PD001128（BlastProDom）及PF00274（HMMPfam）相似的模序,这些序列分子功能均为果糖二磷酸醛缩酶活性。该编码片段亚克隆到栽体上成功构建融合表达质粒pETcFN,转化到大肠杆菌BL21（DE3）中诱导表达,得到分子量为43kDa的融合蛋白。Western blot的结果与预测相符。     

大鼠OB基因克隆及其在大肠杆菌中的表达

采用ＲＴＰＣＲ技术扩增大鼠ＯＢｃＤＮＡ编码区序列。ＰＣＲ产物酶切后定向克隆至ｐＵＣ１９质粒。经核苷酸序列测定表明与文献报道的大鼠ＯＢｃＤＮＡ编码区序列一致。继之构建了ｐＢＶ２２０ｒＯＢ表达质粒并获得了ＯＢ基因在大肠杆菌中的特异表达。大鼠ＯＢ基因产物的获得为研究肥胖与某些非传染性疾病（如糖尿病、高血压病）间的关系提供了条件     

牛肠激酶催化亚基工程菌的构建

目的：构建牛肠激酶催化亚基工程菌。方法：将带有牛肠激酶催化亚基（bEKL）的结构基因进行克隆,将克隆产物纯化后,构建bEKL克隆质粒,后构建bEKL表达质粒,并将表达质粒制导入备感受态细胞,进行蛋白表达。结果：琼脂糖电泳验证牛肠激酶催化亚基基因已经成功地插入表达载体,序列与预期设计一致。结论：表达质粒构建成功。     

Ⅰ型单纯疱疹病毒 gD基因片段的高效表达及抗原性分析

目的 获得高表达的Ⅰ型单纯疱疹病毒(HSV)被膜糖蛋白gD（简称gD1）基因的工程菌。方法 通过计算机分析,筛选出疱疹病毒gD1中优势抗原决定簇的基因片段。将克隆的基因片段插入表达载体pTrxA内,转化大肠杆菌Rosetta,以异丙基-β-D-硫代半乳糖苷诱导表达。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析表达产物。　结果　PCR扩增出约930bp的gD1编码基因目的片段,与预期片段大小相符,经测序鉴定无基因突变;所构建pTrxA-gD1重组表达质粒阳性克隆经PCR与双酶切鉴定,与预期结果一致;含有pTrxA-gD1重组质粒的大肠杆菌Rosetta诱导后得到了高效达,SDS-PAGE显示表达产物约Mr48000（Dalton）。免疫印迹结果表明表达产物具有较好的抗原性。结论　成功构建了pTrxA-gD1表达质粒,实现了成熟gD1蛋白在大肠杆菌中的高效表达,表达产物具有好的抗原性。     

重叠延伸PCR法合成EV71 VP1-LTB融合基因及其原核表达

目的用重叠延伸PCR（overlap extension PCR）法获得人肠道病毒71型vp1与大肠埃希菌不耐热肠毒素B亚单位（ltb）的融合基因,构建EV71 VP1-LTB融合表达质粒并在原核系统表达。方法设计14对引物通过重叠延伸PCR技术合成vp1基因,以ETEC（44815）质粒DNA为模板,PCR扩增ltb基因,将vp1基因与ltb基因连接并测序,连接产物插入原核表达质粒pBEX,构建重组质粒pBEX-VP1-LTB,转化E.coliB1221（DE3）进行表达,SDS-PAGE及W estern blotting分析其表达。电转法将重组质粒转入双歧杆菌。结果合成的vp1基因全长891 bp,测序结果与预期相符,重组表达质粒pBEX-VP1-LTB经PCR及双酶切鉴定,表明构建正确;目的蛋白在E.coliBL21（DE3）中获得了表达,Western bloting分析结果表明该蛋白具有与EV71 VP1抗体的反应原性;电转法成功将重组质粒转入双歧杆菌。结论应用重叠延伸PCR技术成功构建了EV71 VP1-LTB融合表达质粒,并在E.coliBL21（DE3）中获得有生物学功能的VP1表达产物,重组质粒双歧杆菌转化及VP1-LTB融合蛋白的表达研究,为研制EV71分子内佐剂疫苗打下了基础。     

SPF鸡胚中内源性白血病病毒全基因序列鉴定与分析

摘要:以国内某3家SPF鸡场的SPF鸡胚成纤维细胞提取的基因组DNA为模板,参照已发表的序列,设计合成了4对检测内源性白血病病毒引物,分别检测gag基因、pol基因、env基因和LTR片段,结果显示4者检出阳性率很高(gag,29/46;pol,27/46;env,24/46;LTR,31/46).设计合成了8对引物,选取4片段检测均为阳性的样品之一,经PCR成功扩增出了8段连续的、相互部分重叠的目的DNA片段,分别连接入T载体进行克隆测序.用DNAstar软件对测序结果进行拼接,从一个鸡胚得到了内源性白血病病毒前病毒全基因组序列.比较分析发现,该序列env基因与已知的E亚群内源性病毒代表株env基因的核苷酸序列同源性在98.5%以上,全基因组序列同源性在99.1%以上,而与其他亚群代表株同源性相对较低,env基因同源性仅为56.3%～91.5%.     

Cloning and sequence of the crp gene of Escherichia coli K 12.

We have determined the nucleotide sequence of the crp gene of Escherichia coli K 12. From a lambda transducing phage, the crp region was subcloned into pBR322. The gene was localized on the cloned fragment by determining the length of deletions which affect its expression. Its nucleotide sequence was established by using the technique of Maxam and Gilbert. The deduced amino-acid sequence is in agreement with the previously published amino acid composition of the protein (1, 2). Analysis of the sequence confirms that the DNA binding domain is located in the C-terminal portion of the protein.     

内蒙古白绒山羊IGF-IR基因cDNA克隆及组织表达特异性分析

旨在克隆内蒙古白绒山羊IGF-IR基因并分析其基本表达模式.采用RT-PCR克隆基因,将得到的IGF-IR基因cDNA片段的核苷酸序列及其编码的氨基酸序列进行生物信息学分析.半定量RT-PCR进行组织特异性表达检测.获得了内蒙古白绒山羊IGF-IR基因3’端编码区2118 bp的cDNA序列(JN200823),编码705个氨基酸残基.核苷酸序列与牛的IGF-IR( XM606794.3)基因同源性为98％,相应的氨基酸序列同源性为99％.SMART分析表明,推导出的编码蛋白具有跨膜域,酪氨酸激酶催化域.半定量RT-PCR检测表明,IGF-IR基因在绒山羊脑、胰腺、肝、肾组织中均有表达.     

Nucleotide sequence of the insecticidal protein gene of Bacillus thuringiensis strain aizawai IPL7 and its high-level expression in Escherichia coli

A DNA fragment carrying the insecticidal protein gene of Bacillus thuringiensis subsp. aizawai IPL7 was cloned from a 78-kb plasmid. The nucleotide sequence revealed that the cloned DNA fragment contained a 3465-bp protein-coding region with 156-bp 5'-flanking, and 168-bp 3'-flanking regions. The open reading frame encoded a 130,690 Da protein consisting of 1155 amino acid residues. Nucleotide sequence comparison of the aizawai gene with the published berliner 1715 gene showed only 8 nt changes in the coding regions. It was found that 72 bp of the 5'-flanking sequence of the cloned aizawai gene was responsible for constitutive expression of the 130-kDa protein gene in Escherichia coli. The expression was greatly enhanced by introducing the tac promoter upstream from the 72-bp 5'-flanking region of the aizawai gene. Under optimal conditions, the 130-kDa insecticidal protein amounted to 38% of the total cellular protein.     

C型产气荚膜梭菌α毒素基因的克隆与表达

利用PCR技术,从C型产气荚膜梭菌染色体基因组中扩增了1．2kb的α毒素基因,将纯化的PCR产物与载体pGEM-T连接,转化至受体菌JM109中,经NcoI／EcoRI和BamHI／EcoRI酶切鉴定及核苷酸序列测定证实,重组质粒pXCPAl中含有α毒素全基因。随后用NcoI／EcoRI酶切质粒pXCPAl,回收α毒素基因片段,插入到事先经同样酶切处理的载体pET-28c中相应酶切位点,构建了表达质粒pETXAl,经NcoI／EcoRI和BamHI／EcoRI酶切鉴定及核苷酸序列测定证实,表达质粒含有α毒素基因且基因序列和阅读框架正确。重组菌株BL21(DE3)(pETXAl)表达产物经ELISA检测和SDS-PAGE分析,重组菌株表达的α毒素蛋白能够被α毒素单抗识别,其表达量占菌体总蛋白相对含量的16．28％。     

牛肠激酶轻链基因的克隆及其在大肠杆菌中的融合表达

为克隆表达牛肠激酶（enterokinase）轻链(EKL)编码基因,以期应用于融合蛋白的切割与纯化,从市售北方黄牛十二指肠组织中提取总RNA,以RT-PCR方法扩增其cDNA片段,将此片段克隆于pUCmT载体中,经过特异性限制性内切酶酶切分析片段正确后,进行全序列分析。结果表明,克隆的cDNA与GenBank上的序列相比完全一致,得到了编码正确的牛肠激酶轻链基因全序列。随后,将目的基因片段插入pET39b中,构建了融合型表达载体pET39b-EKL,转化大肠杆菌BL21(DE3),用IPTG诱导表达。所获得的pET39b-EKL经过酶切鉴定和测序,证实其插入方向、读码框架正确,所表达重组蛋白经SDS-PAGE分析,相对分子量为65 kDa,表达量达28%,通过镍亲和层析纯化得到融合蛋白DsbA-rEKL的单一条带。该粗酶经脱盐后在适宜的缓冲体系中21℃温育过夜,显示出较高的自催化切割活性,为进一步进行重组牛肠激酶活性的研究及应用奠定了基础。 Abstract:The objective of the study was to obtain the gene of bovine enterokinase light chain,which would be used in the cleavage and purification of fusion proteins．The fragment of bovine enterokinase light chain cDNA was obtained by RT-PCR from a sold bovine′s duodenal mucosa, then cloned into the pUCmT cloning vector and sequenced.Compared with the sequence deposited in GenBank,the cloned gene sequence is correct.Then the interested gene fragment was inserted into the pET39b expression plasmid.The recombinant vector pET39b-EKL was transformed into E.coli BL21(DE3) and induced by IPTG.It was confirmed that the nucleotide sequence was correct on the conjunction site between the recombinant DNA 5′terminal multi-cloning site and recombinant fragment after the analysis of the nucleotide sequence.SDS-PAGE analysis indicated that target product was about 65 kDa which occupied 28% of the total protein.A pure fusion protein was obtained by nickel chelating chromatogram using His*Binding Resin.After desalting and changing buffer,the crude kinase was incubated at 21℃ overnight and demonstrated a high autocatalytic cleavage activity.This investigation would be able to lay a foundation for enterokinase activity research and farther application of expression products on a large scale.     

内蒙古白绒山羊4E-BP1基因cDNA克隆及组织表达特异性分析

旨在克隆内蒙古白绒山羊4E-BP1(真核细胞翻译起始因子4E结合蛋白1)基因并进行生物信息学及表达模式分析。根据已报道物种4E-BP1基因cDNA序列,用primer premier5软件设计引物,通过RT-PCR从绒山羊胎儿成纤维细胞总RNA中扩增出4E-BP1基因编码区cDNA序列,对目的片段进行测序及表达模式分析。克隆到的内蒙古白绒山羊4E-BP1基因cDNA全长357 bp,包含了完整的的ORF,编码118个氨基酸残基。核酸序列与牛、马、人、大鼠及小鼠的同源性分别为98%、90%、90%、88%和87%。4E-BP1基因在绒山羊脑、心脏、睾丸及胰腺组织中均有表达。     

Type 1C fimbriae of a uropathogenic Escherichia coli strain: cloning and characterization of the genes involved in the expression of the 1C antigen and nucleotide sequence of the subunit gene

The genes responsible for expression of type 1C fimbriae have been cloned from the uropathogenic Escherichia coli strain AD110 in the plasmid vector pACYC184. Analysis of deletion mutants from these plasmids showed that a 7-kb DNA fragment was required for biosynthesis of 1C fimbriae. Further analysis of this DNA fragment showed that four genes are present encoding proteins of 16, 18.5, 21 and 89 kDal. A DNA fragment encoding the 16-kDal fimbrial subunit has been cloned. The nucleotide sequence of the structural gene and of the C- and N-terminal flanking regions was determined. The structural gene codes for a polypeptide of 181 amino acids, including a 24-residue N-terminal signal sequence. The nucleotide sequence and the deduced amino acid sequence of the 1C subunit gene were compared with the sequences of the fimA gene, encoding the type 1 fimbrial subunit of E. coli K-12. The data show absolute homology at the N- and C-termini; there is less, but significant homology in the region between the N- and C-termini. Comparison of the amino acid compositions of the 1C and FimA subunit proteins with those of the F72 and PapA proteins (subunits for P-fimbriae) revealed that homology between these two sets of fimbrial subunits is also maximal at the N- and C-termini.     

香蕉果实成熟相关基因ACO1启动子区的克隆及其功能初探

根据已报道的香蕉课实表达ACC氧化酶基因（ACO1）的序列,用改进的接头连接PCR法从香蕉基因组中扩增并克隆了此基因5′旁侧区1526bp的片段,其中包含一个推测的TATA盒序列;与已公布的两个香蕉ACC氧化酶基因启动子序列（分别为934bp和1451bp）的相似性各为97．3％（Lopez-Gomez等）和88．8％（May和Kipp)。将4个含有不同大小启动子区的克隆片段与GUS基因编码区连接构建成嵌合成基因,通过基因枪轰击转入香蕉叶、根和果实的细胞后,瞬时表达结果表明不同大小的ACO1启动子区段都只在果实细胞中指导GUS基因表达,证明该启动子具有指导基因在果实中表达的功能,并推测负责果实特异性的顺式元件可能位于启动子近端0．7kb区段之内,在468至822的355bp区段内可能在与正控制有关的顺式元件。