Research on the Construction of Bacterial Artificial Chromosome Vector DNA and the Potentials in Application

[Objective] The aim of this study was to provide a method for solving the problems in preparing BAC vector with High-copy plasmid pUC119-Bluelox BAC.[Method] With selecting a proper single restriction site,sequences of a single copy BAC vector plasmid were inserted into proper site of High-copy plasmid pUC119 vector.[Result] The gene sequence of BAC vector lost control function of single copy number in new plasmid pUC119-BAC and was copied through High-copy form. The gene sequence of BAC vector basic function was completely cutted off through single enzyme digestion and the control function of single copy could be recovered by auto-connection.[Conclusion] The High-copy pUC119-BAC plasmid was used to copy and amplify high copy of basic function gene sequence in BAC vector,besides that it could be used to construct transfer vector of molecular cloned recombinant virus or BAC library.     

一种用高拷贝质粒载体制备BAC载体基本功能基因的新方法(英文)

[Objective] The aim of this study was to provide a method for solving the problems in preparing BAC vector with High-copy plasmid pUC119-Bluelox BAC.[Method] With selecting a proper single restriction site,sequences of a single copy BAC vector plasmid were inserted into proper site of High-copy plasmid pUC119 vector.[Result] The gene sequence of BAC vector lost control function of single copy number in new plasmid pUC119-BAC and was copied through High-copy form. The gene sequence of BAC vector basic function was completely cutted off through single enzyme digestion and the control function of single copy could be recovered by auto-connection.[Conclusion] The High-copy pUC119-BAC plasmid was used to copy and amplify high copy of basic function gene sequence in BAC vector,besides that it could be used to construct transfer vector of molecular cloned recombinant virus or BAC library.     

Immune Responses in Mice Injected with gD Plasmid DNA of Infectious Bovine Rhinotracheitis Virus

The gene encoding gD of isolate Luojing of infectious bovine rhinotracheitis virus (IBRV)was amplified,sequenced, and cloned into plasmid pcDNA3.1, resulting in a recombinant pcDNA-gD. Groups of BALB/c mice were injected with 100μg of plasmid only or together with liposome. After immunization, serum samples were collected from mice every 2 weeks for a 10-week period and tested for protein-specific antibody with enzyme-linked immunosorbent assay (ELISA). It was showed that the plasmid encoding IBRV glycopretein D developed gene-specific antibody. This report indicates the potential of DNA injection as a method of vaccination.     

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.     

Construction of Prokaryotic Expression Vector of Mouse Nanog Gene and Its Expression

The aim of this study is to construct a prokaryotic expression vector of mouse Nanog gene and to express it in E. coli. A pair of primers was designed according to digestion sites in plasmid pGEX-KG and the Nanog gene sequence published by GenBank. The DNA fragment of 918 bp was amplified by polymerase chain reaction （PCR） from the pNA992 recombinant plasmid with Nanog gene, then cloned into pGEX-KG and transformed into the host E. coli strain TG Ⅰ. The sequence of the fragment was matched with the original sequence of pNA992. It indicated that fusion expression vector, pGEX-KG- Nanog, was constructed successfully. The pGEX-KG-Nanog plasmid was extracted from E. coli strain TG Ⅰ and was transformed into BL21（DE3） for expression. After induction by isopropyl-β-D-thiogalactoside （IPTG） at 37℃, the expression product of Nanog gene was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE）, and the expression condition was optimized. Nanog fusion protein was successfully expressed in the form of inclusion bodies. The molecular weight of the inclusion body was 63 kDa. Meanwhile, the optimum condition for the expression of Nanog fusion protein was induced with 0.8 mmol L^-1 IPTG for 5 h. The mouse Nanog gene was successfully expressed in E. coli, which laid a foundation for the purification of Nanog protein and for the preparation of polyclonal antibody.     

Expression of Aminopeptidase N1 （APN1）, the Main Receptor Protein for Bacillus thuringiensis CrylA Toxin from Helicoverpa armigera Larval Midgut in Trichoplusia ni cells

The aim of this article is to successfully express the Bt （Bacillus thuringiensis） toxin receptor protein located on the internal membrane of larval midgut of cotton bollworm （Helicoverpa armigera Hübner） within eukaryotic expression system, which is one of the key links for clarifying the relationship between receptor and Bt resistance. The fragments of aminopeptidase N1 （APN1） gene without signal peptide in the susceptible and the resistant H. armigera were cloned separately using PCR method, and were separately cloned into pUC 19 vector. After sequencing the gene, the fragments encoding for APN1 without signal peptide were cloned into the Bac-to-Bac baculovirus expression system with transfer vector pFastBacHTB under the polyhedron gene promoter. The recombinant transposing plasmid pFastBacHTB/APN1 was screened and then transformed into Escherichia coli DH10Bac. It was cultured in LB medium, which contained Te, Kan, Ge, X-gal, and IPTG. The resulting recombinant bacmid was transfected into cells of the insect Trichoplusia ni and recombinant baculoviruse was obtained. The lysate of cells infected with recombinant baculoviruse was analyzed by SDS-PAGE and blot analysis. The results showed that the recombinant baculoviruse was fully capable of expressing APN1. The APN1 gene successfully expressed in T. ni cell established the base for continuing the research on its function and relationshio of resistance with Bt.     

弥刈橛斩柿？捎糜诮

jing 100081)The Magnaporthe grisea protein elicitor pemG1 gene was amplified by PCR with the EcoR I and Xho I restriction sites incorporated into the primers,digested by restriction enzymes and ligated with the vector pLexA to construct recombinant bait plasmid pLexA-PEMG1.After the transform of recombinant plasmid into yeast strain EGY48[p8op-lacZ],the autonomous report     

Cloning and Homology Comparison of S Gene for Isolate TH-98 of Porcine Transmissible Gastroenteritis Virus

TH-98 isolate of transmissible gastroenteritis virus (TGEV) was propagated and harvested onswine testicle (ST) monolayer ceil. Two pairs of primers were designed to amplify S gene by RT-PCR accord-ing to the published sequence of TGEV'S gene cDNA with Oligo version 4.1 and DNasis software. The productsof PCR were named Sa and Sb, of 2.3 kb and 2.1 kb respectively. Sa was inserted in EcoR I and Kpn I sitesafter Sb was cloned in Kpn I and Pst I multiple cloning sites of the same pUC18 plasmid. The recombinantpUC-S plasmid was identified and analyzed by corresponding restriction endonuclease and nested PCR on thebasis of the genetic sites of S gene and pUC18 plasmid, which was identified as S gene of TGEV. RecombinantpUC-S was sequenced and analyzed in comparison with the other strains. Gene sequence comparison indicatedthat TH-98 shared 99, 97, 98, 97 and 94% identities with Purdue-115(US), Miller(US), TO14(Japan),FS772(British), 96-1933(British), respectively, their deduced amino acid homology was 99, 97, 97, 96 and93% correspondingly. In addition, the analysis report verified that pUC-S owned a complete open readingframe (ORF) including initiation codon, signal sequences, remaining sequences and termination codon aswell. Therefore, the results affirmed that S gene of TGEV TH-98 was extremely conservative.     

伪狂犬病病毒gG基因缺失通用转移载体的构建

对克隆有伪狂犬病病毒Ea株基因组DNA SphI 15kb片段的质粒pBSA用SphI和KpnI消化，将含gG全基因及其上游PK基因，下游gD基因部分编码区的约2．6kb的片段亚克隆到消除了EcoRI位点的载体pUC19中，获得重组质粒pUSK(E)。以pEGFP-N1为模板，通过PCR扩增约0．3kb的SV40Poly(A)片段，并将其克隆到杆状病毒转座载体pFastBac1的NotI和PstI位点，利用pFastBac1的BamHI和PstI将含有9个酶切位点以及SV40Poly(A)的片段亚克隆到pUSK(E)的相应位点，在插入的同时导致gG基因5‘端编码区约9个酶切位点以及SV40Poly(A)的片段亚克隆到pUSK(E)的相应位点，在插入的同时导致gG基因5‘端编码区的400bp的缺失，构建了由gG启动子驱动gG部分编码区缺失的通用转移载体pgG-Uni。序列分析进一步证实：有7个单一酶切位点可供外源基因直接插入。上述结果为构建以伪狂犬病病毒作载体的多价基因工程疫苗以及利用标记蛋白探讨伪狂犬病病毒在体内的增殖与分布奠定了基础。     

猪传染性胃肠炎病毒S基因杆状病毒/昆虫细胞表达系统重组质粒的构建

用限制性内切酶从克隆质粒pUC—s中回收猪传染性胃肠炎病毒中国分离株,TH-98株完整的病毒保护性抗原基因,即S基因。按照预定的阅读框架,将S基因克隆于供体质粒pFASTBACHTb的EcoRI、PstI多克隆位点上,并进行酶切鉴定,得到阳性重组质粒BAC—S。用DH10BAC对BAC—S进行转位,获得重组质粒pBAC—S。根据S基因酶切位点分析结果及杆状病毒(Bac-ulovirus)转位区结构特点,用相应的限制性内切酶进行酶切鉴定,并用特异性和通用引物进行PCR分析,证明该重组质粒为含有TH-98株完整S基因的可直接在昆虫细胞进行表达的感染性重组质粒。     

含伪狂犬病毒完整UL49基因及部分UL48基因片段的克隆与序列分析

以地高辛标记的伪狂犬病毒（PRV）Ea株UL49.5基因片段为探针，同SalI、KpnI、BamHI消化的PRV Ea株基因组DNA进行Southern杂交，确定SalI2.8kb、KpnI17.0kb、BamHI25.0kb片段中含UL49基因。将SalI2.8kb片段克隆到pUC119中，利用其中的BamHI进行亚克隆，获得重组质粒pUC2.0。测定约2.0kb片段的全序列并进行序列分析，发现该片段包含UL50基因部分编码区，UL49.5基因和UL49基因的完整编码区，并且在UL49基因下游，还存在一段约369个碱基的序列，经与人单纯疱疹病毒I型（HSV－1）、牛单纯疱疹病毒I型（BHV－1）的UL48基因进行比对，具有较高的同源性，推测为PRV UL48基因的部分编码区。     

猪生殖和呼吸综合征中国分离株ORF5 基因的克隆及序列分析

本研究利用对应于PRRSV ATCC VR-2332株及LV株ORF     

甜瓜蔗糖磷酸合成酶(SPS)基因正反义表达载体的构建

为了改善甜瓜果实的品质及研究甜瓜蔗糖磷酸合成酶(SPS)基因的生物学功能,本试验分别构建了甜瓜果实SPS基因的正义及反义表达载体。用PCR方法将克隆到pMD18-T载体上的甜瓜SPS基因用带有KpnⅠ和XbaⅠ酶切位点的引物扩增,然后将该扩增片段克隆到pMD19-T Simple载体上,再用KpnⅠ和XbaⅠ双酶切,得到该基因编码区3.37 kb的cDNA片段,将其定向插入到植物表达载体pROK2的KpnⅠ/XbaⅠ克隆位点,构建了甜瓜果实SPS基因的正义表达载体。将已克隆到pMD18-T载体上的甜瓜SPS基因用KpnⅠ和HincⅡ双酶切,得到该基因编码区830 bp的cDNA片段,将其定向插入到植物表达载体pROK2的KpnⅠ/SmaⅠ克隆位点,构建了甜瓜果实SPS基因的反义表达载体。     

1株猪传染性胃肠炎病毒的分离鉴定及其生物学特性研究

采集1例疑似感染猪传染性胃肠炎病毒(TGEV)仔猪的粪便,运用RT-PCR方法和血清学方法对病料进行TGEV检测,并应用猪睾丸细胞(ST)进行病毒分离培养,分别采用免疫过氧化物酶单层细胞染色法(IPMA)和RT-PCR方法检测病毒在ST细胞中的增殖动态,应用RT-PCR方法扩增分离TGEV S1基因,经序列测定后,采用DNAStar 7.0和Mega 5.0软件对TGEV进行生物信息学分析。结果表明,病料接种到ST细胞培养后,出现明显细胞病变效应(CPE);RT-PCR检测结果显示,TGEV核酸阳性;血清学鉴定TGEV抗原阳性。将分离毒株命名为TGEV-NY。感染12 h后,IPMA检测结果阳性,且可从细胞培养上清中检出TGEV核酸。序列测定分析表明,分离毒株S1基因开放阅读框(ORF)为2 220 bp,编码740个氨基酸。进化分析显示,分离毒株与我国2006年分离的SC-Y毒株亲缘关系最近,分离毒株属于基因Ⅰ群。     

猪传染性胃肠炎病毒实时荧光定量PCR检测方法的建立及应用

针对猪传染性胃肠炎病毒(TGEV)S基因的保守序列设计引物,以TEGV疫苗毒株为模板,克隆S基因,并构建重组阳性质粒,优化反应体系和扩增条件,建立基于SYBR GreenⅠ染料的实时荧光定量PCR检测方法,并对其特异性、重复性和灵敏度进行分析。结果显示:建立的检测方法灵敏度可达10拷贝/μL,标准曲线线性关系良好(r=0.997),扩增效率高,具有良好的特异性和重复性。使用该方法对124份临床疑似TGEV病料进行检测,阳性样本有17份,检测样本的阳性率为13.7%。该方法可用于兽医临床上TGEV的快速检测和流行病学分析。     

含O型FMDVP1-2A、3C基因和EGFP基因表达盒的构建

采用RT-PCR方法分别扩增口蹄疫病毒O/China99毒株的P1-2A和3C基因,将P1-2A基因连接到pUC119载体,3C基因连接到pMD18-T载体,分别得到重组载体pUC119-P1-2A和pMD18-T-3C;将重组载体pUC119-P1-2A用HindⅢ、BamHⅠ酶切,重组载体pMD18-T-3C用BamHⅠ、NheⅠ酶切;利用酶切所得到的基因片段P1-2A、3C有共同的BamHⅠ酶切位点,实现基因P1-2A、3C的连接,构建重组载体pMD18-T-P1-2A-3C.将基因P1-2A-3C与启动EGFP表达的双向串连痘苗病毒启动子P7.5相连,构建载体p-EGFP-N1-P7.5-P1-2A-3C,并进行PCR扩增、酶切鉴定及序列测定.结果表明:试验成功构建了一侧启动表达P1-2A-3C基因,一侧启动表达标记基因EGFP的表达盒p-EGFP-N1-P7.5-P1-2A-3C.     

重组马立克病病毒CVI988/Rispens的构建

根据Ⅰ型马立克病病毒（MDV）强毒GA株基因序列,设计两对引物,用PCR方法扩增出CV1988／Rispens株的US10及其侧翼序列,分别克隆入pUC18载体中。经测序检测正确后,进一步插入含CMV启动子的绿色荧光蛋白（EGFP）基因表达盒,获得了含EGFP报告基因的转移载体质粒pPUC18-US10-EGFP。通过同源重组,成功地筛选出表达EGFP的重组病毒rCV1988-EGFP,经传代证明重组rCV1988-EGFP在感染的CEF细胞中能稳定表达EGFP。结果表明：构建的重组转移载体质粒正确,US10是MDV复制非必需片段,为进一步利用US10区构建重组MDV多价基因工程疫苗奠定了基础。