重叠PCR-酶切连接法人工合成风疹病毒E1基因及其重组质粒构建

目的:利用重叠PCR-酶切连接法人工合成风疹病毒E1基因的全长序列.方法:对风疹E1基因进行生物信息学分析,根据大肠杆菌密码子偏爱性对其密码子进行优化;设计多对寡核苷酸引物,以重叠PCR法分别合成该基因的3个片段,测序鉴定后以酶切连接法将各段拼接成全长为1 443 bp的RV rE1,将E1基因的全长序列克隆导入原核表达载体pET32a.结果:分别进行8轮、5轮和6轮的重叠PCR扩增,合成风疹基因3个片段;以酶切连接法将3个片段拼接成全长rE1基因并克隆入pET32a构建成载体pET32-RV rE1,PCR、酶切和测序鉴定结果表明,合成的E1基因大小、序列与预期相符;构建获得重组质粒pET32-RV rE1.结论:成功合成了密码子优化的风疹病毒E1基因并构建其重组质粒pET32-RV rE1.     

αB-晶状体蛋白CRYAB基因重组表达载体的构建及分子生物学鉴定

目的:构建人晶状体蛋白CRYAB基因与真核表达载体pIRES2-DsRed-Express的重组体,为研究人晶状体蛋白CRYAB基因的功能奠定基础.方法:根据人晶状体蛋白CRYAB基因的核苷酸序列,设计并合成分别带有酶切位点的引物,从pMD 18T-CRYAB基因克隆载体中扩增出CRYAB基因外显子片段,与载体pIRES2-DsRed-Express连接构建人晶状体蛋白CRYAB基因的表达载体,转化入大肠杆菌DH5α中.经抗生素筛选阳性克隆,通过酶切图谱分析、菌落PCR和测序鉴定所构建的表达载体.结果:构建的载体经PCR、酶切鉴定和测序证实插入方向正确,表达阅读框正确,载体构建成功.结论:重组人晶状体蛋白CRYAB基因表达载体的构建为研究CRYAB基因的功能及进一步研究先天性白内障的发病机制奠定了基础.     

MTERF1基因真核表达载体的构建及其在C-33A细胞中的表达研究

采用PCR技术从pGEM-MTERF1重组克隆载体中扩增出人MTERF1基因cDNA的开放阅读框序列,经KpnⅠ和XhoⅠ双酶切后,以pcDNA3.1(+)为真核表达载体,构建重组质粒pcDNA3.1(+)-MTERF1;通过PCR、双酶切和DNA测序方法对重组子进行鉴定;将重组真核表达质粒pcDNA3.1(+)-MTERF1转染C-33A细胞,利用免疫印迹法检测MTERF1蛋白的表达。结果显示,重组质粒pcDNA3.1(+)-MTERF1经双酶切鉴定和菌落PCR鉴定均获得大小为1 200 bp的目的条带,DNA测序结果表明该序列与GenBank中人MTERF1基因序列完全相同,插入基因的大小和方向正确;免疫印迹结果表明,MTERF1蛋白的表达水平在转染pcDNA3.1(+)-MTERF1的C-33A细胞中表达高于转染pcDNA3.1(+)的C-33A细胞。通过对人MTERF1基因的真核表达载体的构建,为进一步研究人MTERF1基因的功能奠定了基础。     

人RIP2基因启动子绿色荧光蛋白表达载体的构建与鉴定

目的:构建人RIP2基因启动子的绿色荧光蛋白表达载体。方法:根据特定限制性内切酶位点,以人基因组DNA为模板,PCR扩增含人RIP2基因启动子不同长度2段序列,构建含人RIP2基因启动子驱动的绿色荧光蛋白载体pEGFP-C2-RIP2(750 bp)wt、pEGFP-C2-RIP2(941 bp)wt,用VspⅠ和NheⅠ双酶切鉴定重组质粒,进行DNA序列分析,重组质粒经阳离子聚合物JetPeiTM介导转染HEK293细胞48 h后观察。结果:酶切鉴定和序列测定证实目的基因已插入重组质粒;细胞转染结果表明,重组质粒转染HEK293细胞均能表达绿色荧光,其中构建的pEGFP-C2-RIP2(750 bp)wt重组质粒绿色荧光表达强于pEGFP-C2-RIP2(941 bp)wt。结论:成功构建2段不同长度的人RIP2基因启动子绿色荧光蛋白表达载体。     

IFI16基因反义干扰表达载体的构建与鉴定

为了构建可在人喉癌细胞中稳定表达 IFI16基因短发夹RNA（shRNA）的表达载体,设计合成的IFI16基因shRNA片段,连接到经BamH I和 EcoR I双酶切的pGreenPuroTM shRNA表达载体中,连接产物转化大肠杆菌后挑取几个抗性菌落,用PCR技术初步进行鉴定,经PCR初步鉴定为重组质粒的一个重组子DNA用测序进一步鉴定,测序结果显示成功构建了 IFI16基因shRNA的表达载体pGreenPuro-IFI16 shRNA 。     

结核分枝杆菌Ag85B基因克隆及真核表达质粒pIRES-Ag85B的构建

目的:克隆并构建编码结核分枝杆菌Ag85B分泌蛋白的重组真核表达质粒,为进一步研究其在开发新型的结核病疫苗和结核病免疫诊断试剂奠定了基础。方法:采聚合酶链反应(PCR)方法从结核分枝杆菌H37Rv基因组中扩增出Ag85B基因,然后用双内切酶消化PCR产物和pIRES质粒,用T4 DNA连接酶连接后,转化入感受态E.coli DH5a,产物铺AmpR抗性的平板,阳性克隆用酶切和DNA测序鉴定并进入blast网页比对测序的序列。结果:酶切鉴定所切下的片段大小与预计相符,测序比对后结果与目的基因完全一致,证实符合表达框架。结论:成功地克隆并构建Ag85B基因的真核重组表达质粒pIRES-Ag85B。     

黑曲霉F246的phyA基因克隆及其新型表达载体构建

利用PCR法直接从自行筛选、鉴定的黑曲霉F246中扩增出phyA基因,再将PCR产物重组于乳酸菌表达载体pMG36e,构建phyA基因的新型表达载体pMG36e-phyA.以黑曲霉F246基因组为模板,对phyA基因的成熟肽(1 347 bp)进行PCR扩增.再将PCR产物克隆入pMD18T质粒,经DNA测序和氨基酸分析、鉴定正确后,亚克隆入乳酸菌表达质粒pMG36e,构建工程菌Lactobacillus MG1363-pMG36e-phyA.重组乳酸菌表达载体pMG36e-phyA的构建,可望获得大量、高活性植酸酶,为研制多功能微生态制剂奠定基础.     

neuritin基因杆状病毒表达载体的构建

为研究Neuritin蛋白的功能,将neuritinORF在杆状病毒-昆虫表达系统中表达,构建杆状病毒表达载体.以308D10为模板,利用PCR方法扩增neuritinORF,定向克隆法将其克隆至PFASTBAC-HTA转移载体中.然后利用同源重组原理将其转移至杆粒bacmid中.得到携带neuritin ORF的重组杆粒.本实验获得了重组目的基因真核表达载体并经PCR鉴定,插入片段方向、大小正确,DNA测序分析表明插入处接头和读框与预期序列相符,成功构建了Neuritin的杆状病毒表达载体.     

原核表达HIV-1抗原蛋白的ELISA活性检测

研究了HIV-1 gp120s和gp41在大肠杆菌中表达产物的抗原特异性.首先构建表达质粒p120s和pT41,并转化到BL21和M15中.宿主细胞经IPTG诱导表达重组蛋白,SDS-PAGE和Western blot表征分析蛋白产物.总蛋白中gp120s占17.9%,为可溶性表达;gp41占24.6%,为包涵体形式.亲和层析法检测蛋白纯度分别为63.1%和89%.酶联免疫吸附(ELISA)实验说明重组蛋白有良好的抗原特异性.     

The interaction between the membrane-proximal external region and the N-trimer region of HIV-1 gp41: Involvement in viral fusion

The membrane proximal external region (MPER) of gp41 is extremely conserved among diverse HIV-1 variants, implying its important role in viral infection. Interestingly, two of the most broadly neutralizing antibodies, 2F5 and 4E10, specifically recognize this region. Our previous study demonstrated that the antigenicity and immunogenicity of 4E10 epitope are affected by remodeling gp41 fusion core, suggesting that the MPER may be associated with gp41 core and involved in gp41-mediated membrane fusion. Here we measured the binding activity of 4E10 epitope peptide (D4E10P) with various gp41 core-derived peptides and found that the N-trimer region in a construct designated N-trimer-6HB interacted significantly with D4E10P. Using N-trimer-6HB to screen a phage library, we identified a motif (WF) located in 4E10 epitope that may play a certain role in the interaction of gp41 MPER with the N-trimer in gp41 fusion core and, we thus speculated upon the potential involvement of MPER in the fusion process between viral envelope and target cell membrane. Supported by National Key Basic Research and Development Program of China (Grant No. 2007CB914402)     

链霉菌H197 mtg基因毕赤酵母表达载体的构建与鉴定

将酵母胞内表达质粒pPIC3.5k重组成带有谷氨酰胺转胺酶的新型表达载体pPIC3.5k/MTG,为谷氨酰胺转胺酶（MTG）在毕赤酵母中的表达研究提供了实验基础.利用PCR的方法扩增出MTG基因片段,将目的片段和质粒pPIC3.5k进行双酶切后进行连接,构成重组质粒,然后转化至E.coli DH5α感受态细胞中,筛选阳性克隆,经测序证明为目的基因片段.结果表明：经过PCR和酶切均证明已经将目的片段转到酵母表达载体pPIC3.5k内.     

福氏志贺菌ipaB基因的克隆及序列测定

目的:克隆福氏志贺菌(Shigella flexneri,S.flexne)的ipaB基因。方法:以福氏志贺菌2a 2457T(Nal)r为模板,用PCR扩增ipaB目的基因片段,克隆至pQE-30表达载体中,构建含目的基因的表达质粒pQE-ipaB。结果:构建了重组质粒pQE-ipaB,ipaB基因全长1 743 bp,经测序分析与预期相符。结论:成功克隆了ipaB基因。     

Predefined GPGRAFY-Epitope-Specific Monoclonal Antibodies with Different Activities for Recognizing Native HIV-1 gp120

A seven-amino acid epitope GPGRAFY at the tip of the V3 loop in HIV-1 gp120 is the principal neutralizing epitope, and a subset of anti-V3 antibodies specific for this epitope shows a broad range of neutralizing activity. GPGRAFY-epitope-specific neutralizing antibodies were produced using predefined GPGRAFY-epitope-specific peptides instead of a natural or recombinant gp120 bearing this epitope. All six monoclonal antibodies (mAbs) could recognize the GPGRAFY-epitope on peptides and two of the antibodies, 9D8 and 2D7, could recognize recombinant gp120 in enzymelinked immunosorkentassy (ELISA) assays. In the flow cytometry analysis, the mAbs 9D8 and 2D7 could bind to HIV-Env CHO-WT cells and the specific bindings could be inhibited by the GPGRAFY-epitope peptide, which suggests that these two mAbs could recognize the native envelope protein gp120 expressed on the cell membrane. However, in syncytium assays, none of the mAbs was capable of inhibiting HIV-Env-mediated cell membrane fusion. The different activities for recognizing native HIV-1 gp120 might be associated with different antibody affinities against the epitopes. The development of conformational mimics of the neutralization epitope in the gp120 V3 loop could elicit neutralizing mAbs with high affinity.     

Biological function of HIV-1 transmembrane protein gp41──A study on a putative cellular receptor of gp41

Since 1992, the study of biological functions of HIV-1 gp41 has made great progress. Experimental evidence from several research groups demonstrated that gp41 has a putative cellular receptor. A recombinant soluble gp41 (aa539-684) and gp41 immunosuppressive peptide (aa583-599) could bind to human B lymphocytes and monocytes, but weakly bind to T lymphocytes. It was found that gp41 contains two cellular binding sites (aa583-599 and 641-675). GP41 could selectively inhibit cell proliferation of human T, B lymphocytes and monocytes, enhance human MHC class Ⅰ, Ⅱ and ICAM-1 molecule expression on cell surface. Gp41 binding proteins and a monoclonal antibody against the first binding site could inhibit this modulation effect. Amino acid sequence homology exists between gp41 and human type Ⅰ interferons, and the homologous region is located in the first binding site on gp41 and in the receptor binding site on type Ⅰ interferons. Studies in other groups indicate that both binding sites in gp41 may be associated with HIV infection of cells. Peptides containing two binding sites could respectively inhibit HIV infection of cells. A monoclonal antibody recognizing the second binding site could neutralize lab-strains and recently separated strains of HIV-1. Besides, antibodies against two regions (homologous with gp41 binding sites) of SIV transmembrane protein gp32 could protect macaques from SIV infection. These results suggest that the study of gp41 binding sites and cellular receptor could contribute to understanding the mechanism of HIV infection and to developing HIV vaccine and anti-HIV drugs.     

Characterization of Antibody Responses Against the 2F5 Epitope ELDKWA sing HIV-1 Env-Mediated Membrane Fusion and Neutralization Assays

The epitope ELDKWA, which is located in the membrane-proximal external region (MPER) of HIV-1 gp41, is an important neutralizing epitope. The human monoclonal antibody (mAb) 2F5 against this epitope shows broad neutralizing activity toward many HIV strains. However, several reports have shown that the epitope-specific mAbs induced by peptides containing MPER did not exhibit the same neutralizing activities as human mAb 2F5. In this study, four ELDKWA epitope specific mAbs (9E7, 7E10, 6B5, and 2B4) induced by immunization with the ELDKWA epitope in varied molecular contexts, all showed inhibitory activities with different potencies in HIV-1 Env-mediated membrane fusion assays and pseudovirus neutralization assays. This result indicates that though these antibodies recognize the epitope ELDKWA, their characterizations differ from that of neutralizing antibodies, implying that the neutralizing mAbs can be induced but also need to be screened, and the protective ability of a related vaccine antigen depends on the concentration of the neutralizing mAbs in the induced polyclonal antibodies.     

水稻磷酸酯酶2A基因蛋白的原核表达载体构建、表达和纯化

利用PCR技术,从水稻品种日本晴幼穗cDNA中扩增磷酸酯酶2A的基因ORF片段,并将其克隆入原核表达载体pGEX-6p-1中构建重组质粒pGEX-6p-1-PP-2A,经限制性内切酶酶切鉴定以及测序结果表明成功构建了原核表达载体pGEX-6p-1-PP2A。转化E.coli JM109并通过终浓度为0．4mmol／L的IPTG诱导,表达出39kD的CST-PP2A融合蛋白,并用蛋白亲和层析柱GSTrap FF对表达产物进行纯化,为下一步的研究打下了实验基础。     

Broad neutralization coverage of HIV by multiple highly potent antibodies

Broadly neutralizing antibodies against highly variable viral pathogens are much sought after to treat or protect against global circulating viruses. Here we probed the neutralizing antibody repertoires of four human immunodeficiency virus (HIV)-infected donors with remarkably broad and potent neutralizing responses and rescued 17 new monoclonal antibodies that neutralize broadly across clades. Many of the new monoclonal antibodies are almost tenfold more potent than the recently described PG9, PG16 and VRC01 broadly neutralizing monoclonal antibodies and 100-fold more potent than the original prototype HIV broadly neutralizing monoclonal antibodies. The monoclonal antibodies largely recapitulate the neutralization breadth found in the corresponding donor serum and many recognize novel epitopes on envelope (Env) glycoprotein gp120, illuminating new targets for vaccine design. Analysis of neutralization by the full complement of anti-HIV broadly neutralizing monoclonal antibodies now available reveals that certain combinations of antibodies should offer markedly more favourable coverage of the enormous diversity of global circulating viruses than others and these combinations might be sought in active or passive immunization regimes. Overall, the isolation of multiple HIV broadly neutralizing monoclonal antibodies from several donors that, in aggregate, provide broad coverage at low concentrations is a highly positive indicator for the eventual design of an effective antibody-based HIV vaccine.     

大鼠组织型金属蛋白酶抑制剂-1在毕赤酵母中的重组表达

提取SD大鼠肝组织总RNA,通过RT-PCR扩增TIMP-1 cDNA片段.将扩增产物克隆至pUCm-T载体上,PCR、限制性酶切及测序证实后,EcoRI和NotI双酶切pUCm-T-TIMP-1重组体,回收TIMP-1,再将其亚克隆到酵母表达载体pPIC9K中,并将阳性克隆进行PCR、酶切和测序分析,构建pPIC9K-TIMP-1表达载体,电击转化到毕赤酵母,通过表型筛选和诱导表达得到蛋白表达工程菌,为进一步研究TIMP-1功能和作用机理等的研究创造了条件.     

Soluble CD4 molecules neutralize human immunodeficiency virus type 1

Human immunodeficiency virus (HIV) infection can bring about total collapse of the immune system by infecting helper T lymphocytes which express CD4, the molecule which mediates interaction between the cell surface and viral envelope glycoprotein gp120 (refs 3-10). HIV apparently escapes the effects of neutralizing antibodies in vivo by generating new variants which must still interact with CD4 to maintain a cycle of infection. One route to block HIV infection, therefore, could use solubilized CD4 protein to inhibit attachment of the virus to its target cell. We have used recombinant DNA techniques to generate soluble forms of CD4, and show here that these are potent inhibitors of HIV infection in vitro.