His-猪生长激素融合基因在Bm-N细胞和蚕体内的表达与纯化

用构建的带有His—Tag pgh融合基因的重组杆状病毒Bm—BacPAK6—pgh研究了Bm—BacPAK6—pgh在家蚕细胞(Bm—N)和蚕体内的表达，并对蚕体表达产物进行了纯化．SDS—PAGE电泳分析显示，重组病毒Bin—BacPAK6—pgh在Bin—N细胞、蚕体中得到了融合表达，Western blot分析表明，Bm—N细胞、蚕体中表达的融合蛋白与大肠杆菌表达的猪生长激素具有相同的抗原性．Bm-BacPAK6-pgh在Bin—N细胞内的表达始于24h，而蚕体中的表达始于72h，二者的表达峰分别在96h和120h．经40％饱和度硫酸铵盐析和Ni—NTA Agarose亲和柱二步纯化可获得SDS—PA(正电泳纯的具有抗原性的重组猪生长激素融合蛋白。     

血小板生成素在毕赤酵母中的表达

以人胎肝cDNA文库为模板,用PCR和DNA重组技术,将TPOcDNA克隆到pGEM     

Immune response in cattle inoculated with the recombinant complete polyprotein of foot-and-mouth disease virus from Bombyx mori larvae

The intact open reading frame (ORF) of foot-and-mouth disease virus (FMDV) Asia I/XJ strain was am- plified by RT-PCR and inserted into the transfer vector pVL1393 to generate plasmid pVL-ORF. Bm-N cells were transfected with pVL-ORF and linearized Bm-BacPAK6 DNA, and the recombinant silkworm baculovirus Bm-ORF containing the full ORF of FMDV was obtained. The results of indirect im- munofluorescence assay (IFA) showed that Bm-ORF could be expressed efficiently in Bm-N cell. After inoculating the early 5th instar larvae of silkworm, the polyprotein of FMDV could be detected by sandwich ELISA and empty capsid-like particles could be observed under the electron microscope. Expression products from silkworm were used as the antigen to immunize the cattle. The specific an- tibody was induced in all vaccinated animals. The immunized cattle were challenged with the virulent FMDV Asia I/XJ strain, two of the four cattle were completely protected and clinical symptoms were alleviated and delayed in the others. The results suggest that this strategy might be used to develop the new subunit FMDV vaccine.     

猪传染性胃肠炎病毒S基因B和C抗原位点片段在昆虫细胞中的胞内表达

通过基因重组的方法,在昆虫细胞胞内表达了猪传染性胃肠炎病毒（TGEV）S基因B和C抗原位点片段.表达蛋白经Dot-ELISA检测具有良好的抗原性.本研究为TGEV的血清学检测方法的建立提供了必要的物质基础.     

Cloning and expression of HIV-1 p24 gene in insect cells by using BAC-TO-BAC system

Recombinant transposing vector pFHIV24 was constructed by cloning the HIV-1 p24 gene into the Multiple cloning site (MCS) of the transposing vector pFastBac1 in the correct orientation with respect to the polyhedrin promoter. Recombinant bacmid bHIV24 was obtained by transposing a mini-att Tn7 element from the recombinant pFHIV24 to the mini-att Tn7 attachment site on the bacmid by Tn7 transposition functions provided by the helper plasmid. Minipreparation of recombinant bacmid DNA was transfected intoSpodoptera frugiperda (Sf9) cells to get the recombinant virus. Fresh insect Sf9 cells were infected with the recombinant virus containing p24 to express the target protein. The target protein expressed was analyzed on a 15% polyacrylamide gels and then used as antigen to check HIV-1 positive serum by ELISA. Our positive result shows that the expressed p24 protein could be used as standard antigen for HIV-1 diagnosis by ELISA and other reliable diagnostic methods of HIV-1 infection. Supported by the World Bank Boan Program Mallam Nock Joshua: born in 1967, Master of Science To whom correspondence should be addressed (027-7882712-2938)     

Cloning and expression of HIV-1 p24 gene in insect cells by using BAC-TO-BAC system

Recombinant transposing vector pFHIV24 was constructed by cloning the HIV-1 p24 gene into the Multiple cloning site (MCS) of the transposing vector pFastBac1 in the correct orientation with respect to the polyhedrin promoter. Recombinant bacmid bHIV24 was obtained by transposing a mini-att Tn7 element from the recombinant pFHIV24 to the mini-att Tn7 attachment site on the bacmid by Tn7 transposition functions provided by the helper plasmid. Minipreparation of recombinant bacmid DNA was transfected intoSpodoptera frugiperda (Sf9) cells to get the recombinant virus. Fresh insect Sf9 cells were infected with the recombinant virus containing p24 to express the target protein. The target protein expressed was analyzed on a 15% polyacrylamide gels and then used as antigen to check HIV-1 positive serum by ELISA. Our positive result shows that the expressed p24 protein could be used as standard antigen for HIV-1 diagnosis by ELISA and other reliable diagnostic methods of HIV-1 infection. Supported by the World Bank Boan Program Mallam Nock Joshua: born in 1967, Master of Science To whom correspondence should be addressed (027-7882712-2938)     

Human hepatitis B vaccine from recombinant yeast

The worldwide importance of human hepatitis B virus infection and the toll it takes in chronic liver disease, cirrhosis and hepatocarcinoma, make it imperative that a vaccine be developed for worldwide application. Human hepatitis B vaccines are presently prepared using hepatitis B surface antigen (HBsAg) that is purified from the plasma of human carriers of hepatitis B virus infection. The preparation of hepatitis B vaccine from a human source is restricted by the available supply of infected human plasma and by the need to apply stringent processes that purify the antigen and render it free of infectious hepatitis B virus and other possible living agents that might be present in the plasma. Joint efforts between our laboratories and those of Drs W. Rutter and B. Hall led to the preparation of vectors carrying the DNA sequence for HBsAg and antigen expression in the yeast Saccharomyces cerevisiae. Here we describe the development of hepatitis B vaccine of yeast cell origin. HBsAg of subtype adw was produced in recombinant yeast cell culture, and the purified antigen in alum formulation stimulated production of antibody in mice, grivet monkeys and chimpanzees. Vaccinated chimpanzees were totally protected when challenged intravenously with either homologous or heterologous subtype adr and ayw virus of human serum source. This is the first example of a vaccine produced from recombinant cells which is effective against a human viral infection.     

血小板生成素在枯草杆菌中的表达分泌及前肽作用

利用PCR方法扩增得到枯草杆菌本身的表达调控序列SP（含有信号肽）和Pro序列（包含信号肽和前肽）,分别与血小板生成素(TPO)结构基因及枯草杆菌载体片段连接,构建两个重组TPO枯草杆菌表达载体,转化受体菌,得到DB403（SP-TPO）和DB403（Pro-TPO）重组克隆,比较有无前肽对其表达分泌的作用.用ELISA和Western Blot检测TPO的表达分泌,发现DB403（Pro-TPO）能够表达分泌TPO,而DB403（SP-TPO）未发现TPO的分泌.对小鼠腹腔注射DB403（Pro-TPO）发酵浓缩液,发现能够明显增加血小板数目,与自身对照相比增加84.2％,与生理盐水对照比较增加99.7％.以上实验说明Pro-TPO能够表达分泌有明显活性的TPO蛋白,而且前肽对外源蛋白的分泌是必要的.     

钩端螺旋体外膜脂蛋白LipL32基因的克隆和表达及其在ELISA检测中的应用

钩端螺旋体 (钩体 )病是一种全球性的人兽共患病。人类感染钩端螺旋体后表现出多种器官和系统病变 ,愈后不良 ;也可垂直传播感染胎儿 ,引起流产。动物中 ,犬钩端螺旋体的感染率较高 ,感染后 ,临床表现多样 ,大多呈隐性感染 ,不发病 ,但钩体在肾脏中长期存在 ,持续随尿液向外排菌。由于钩端螺旋体对人的强致病性 ,以及由动物传播给人引起的严重公共卫生问题 ,因此快速准确地进行钩端螺旋体病的诊断 ,显得尤为重要 ,也是目前钩端螺旋体研究中亟待解决的问题。钩端螺旋体属钩端螺旋体科 ,钩端螺旋体属问号状钩端螺旋体种的成员。目前发现血清型…     

重组猪生长激素抗体的制备与纯化

为了获得重组猪生长激素抗体,对重组猪生长激素融合基因的真核表达产物进行免疫印迹(Western blot)检测,将利用DNA重组技术构建的重组质粒pPGH020在大肠杆菌(E.coli)BL21中进行诱导表达.表达产物经Ni+亲和层析柱纯化,获得纯化的重组猪生长激素融合蛋白.然后以此为抗原免疫新西兰大白兔,获得多克隆抗体,抗体再经硫铵沉淀、透析和亲和层析纯化.Western印迹结果表明,该纯化抗体显示了良好的免疫反应,其灵敏度比兔抗rpGH抗血清提高50倍,为猪生长激素融合基因在真核细胞的表达及功能鉴定奠定了基础.     

Production of human alpha-interferon in silkworm using a baculovirus vector

Microorganisms are generally used for mass production of foreign gene products, but multicellular organisms such as plants have been proposed as an economical alternative. The silkworm may be useful in this context as it can be cultured easily and at low cost. We have therefore developed a virus vector to introduce foreign genes, for example, the gene for human alpha-interferon (IFN-alpha), into silkworms. We used the baculovirus Bombyx mori nuclear polyhedrosis virus (BmNPV) which has a large (greater than 100 kilobases, kb) double-stranded circular DNA genome within its rod-shaped capsid. Baculoviruses have been used previously as vectors for expression of beta-interferon and beta-galactosidase in established cell lines. Although BmNPV has not been used previously as an expression vector, it has an advantage over the baculovirus Autographa californica NPV in that it has a narrower host range and will not grow in wild insect pests in the field. In the present study, the polyhedrin gene encoding the major inclusion body protein of BmNPV was identified by hybridization with complementary DNA and cloned in a plasmid. For insertion of foreign genes, we constructed a recombinant plasmid carrying a polylinker linked to the promoter of the polyhedrin gene, and inserted the IFN-alpha gene into this plasmid. The resulting plasmid and the BmNPV genomic DNA were co-transfected into BM-N cells, and stable recombinant viruses isolated by plaque assay on BM-N cells. The recombinant virus replicated in silkworm larvae, which synthesized as much as 5 X 10(7) units (approximately 50 micrograms) of interferon in their haemolymph.     

Expression of green fluorescent protein gene with baculovirus vector in insect cells

The green fluorescence of bioluminescent jellyfishAequorea victoria is due to the presence of the green fluorescent protein (GFP). To examine whether the GFP gene can be applied as a reporter gene in insect cells, a baculovirus transfer vector containing the neomycin resistance gene (neo) was established. The GFP gene was subcloned into the vector downstream of the polyhedrin gene (ocu) promoter. In the presence of G418, the recombinant virus can be purified. Expression of the GFP gene in the recombinant virus should give rise to synthesis of the GFP with a molecular weight of 30×10³ dalton, and is observable by the strong green light irradiated by ultraviolet or blue light in viable intact insect cells. The GFP produced in insect cells has typical fluorescent spectra indistinguishable from those of the purified native GFP. The GFP gene as a good reporter gene can be applied to the baculovirus-insect cell expression system. Supported by the National Natural Science Foundation of China Hu Jianhong: born in July, 1972, Master graduate student     

禽流感病毒H5N1血凝素基因和神经氨酸酶基因在真核酵母菌中的表达

目的克隆、表达和鉴定禽流感病毒H5N1血凝素基因（hemagglutinin,HA）和神经氨酸酶基因（neuramidinase,NA）序列,为制备抗体和基因工程疫苗打下基础。方法在成功克隆禽流感病毒H5N1全长HA、NA基因并测序的基础上,将部分基因序列克隆到表达载体pMET A上,构建了重组表达质粒pMET A/HA（49～1 587 bp）、pMET A/NA（121～1 200 bp）,电转化真核酵母菌pMAD16,甲醇诱导表达,利用Ni2＋亲和层析柱对重组蛋白进行纯化,并用Western Blotting和ELISA方法检测其抗原性。结果重组蛋白在酵母菌中可以高效表达,SDS-PAGE显示蛋白表达后形成了二聚体,蛋白纯度占总蛋白的95%以上,ELISA和Western Blotting实验证实,重组蛋白具有良好的抗原性。结论本研究成功克隆和表达了禽流感病毒H5N1 HA、NA基因序列,为禽流感病毒H5N1诊断试剂和疫苗的开发等进一步的研究提供了依据。     

人细胞色素P450氧化还原酶的克隆表达和抗体的制备

细胞色素P450还原酶(CPR)是人细胞色素P450酶系的电子供给者,对后者活性的发挥起到重要作用.本研究将从人胚胎cDNA文库中得到的人CPR的编码基因,连接入pT7450表达载体中,在大肠杆菌BL21(DE3)中高效表达并纯化.每升发酵液可得到纯化蛋白49 mg,占总蛋白含量的10%,以细胞色素C为底物检测酶活性,比活为65 u/mg.利用纯化的CPR作为抗原,常规免疫纯系大耳家兔,获得高效价、高特异性的抗血清,抗体滴度为1∶200 000(ELISA法),纯化后抗体应用于不同组织中CPR含量的评价,证明重组CPR及其抗体可用于细胞色素P450的体外药物代谢及细胞色素P450与CPR作用机理的研究.     

人工饲料育家蚕/杆状病毒表达猪瘟病毒囊膜糖蛋白E2基因

以重组病毒Bm-BacPAK6-E2接种全龄人工饲料育及5龄桑叶育家蚕皓月×菁松幼虫、蛹,观察重组病毒的感染发病情况,调查病蚕、蛹的采血量并进行SDS-PAGE分析.结果表明,全龄人工饲料育家蚕接种重组病毒的成功率、采血量等指标均与5龄桑叶育相近,SDS-PAGE蛋白质电泳在家蚕幼虫、蛹血淋巴中均检测到1条与目的蛋白理论分子量(63 kD)相符的特异性蛋白条带,初步认为是重组病毒Bm-BacPAK6-E2的融合表达产物.     

HIV-1C亚型DNA疫苗诱导小鼠免疫反应的研究

 构建含中国流行株HIV-1 C亚型核心蛋白gag基因的重组质粒pVAX-gag,并在体外进行了表达与鉴定.同时构建了含此gag基因的原核表达质粒pGEX-gag,表达纯化并鉴定重组蛋白Gag.以质粒pVAX-gag免疫Balb/C小鼠后,用ELISpot和流式细胞仪检测其细胞免疫反应.再以纯化后的重组蛋白Gag作为包被抗原,用ELISA检测其体液免疫反应.结果显示重组质粒pVAX-gag免疫小鼠后可有效地诱导机体产生细胞免疫和体液免疫反应,且免疫剂量和免疫效果存在一定的正相关性.重组原核表达质粒pGEX-gag的表达产物能与抗p24单克隆抗体发生特异性反应,可用于抗HIV抗体检测.     

The molecular organization of cypovirus polyhedra

Cypoviruses and baculoviruses are notoriously difficult to eradicate because the virus particles are embedded in micrometre-sized protein crystals called polyhedra. The remarkable stability of polyhedra means that, like bacterial spores, these insect viruses remain infectious for years in soil. The environmental persistence of polyhedra is the cause of significant losses in silkworm cocoon harvests but has also been exploited against pests in biological alternatives to chemical insecticides. Although polyhedra have been extensively characterized since the early 1900s, their atomic organization remains elusive. Here we describe the 2 A crystal structure of both recombinant and infectious silkworm cypovirus polyhedra determined using crystals 5-12 micrometres in diameter purified from insect cells. These are the smallest crystals yet used for de novo X-ray protein structure determination. We found that polyhedra are made of trimers of the viral polyhedrin protein and contain nucleotides. Although the shape of these building blocks is reminiscent of some capsid trimers, polyhedrin has a new fold and has evolved to assemble in vivo into three-dimensional cubic crystals rather than icosahedral shells. The polyhedrin trimers are extensively cross-linked in polyhedra by non-covalent interactions and pack with an exquisite molecular complementarity similar to that of antigen-antibody complexes. The resulting ultrastable and sealed crystals shield the virus particles from environmental damage. The structure suggests that polyhedra can serve as the basis for the development of robust and versatile nanoparticles for biotechnological applications such as microarrays and biopesticides.     

抗棉铃虫组织蛋白酶B单克隆抗体的制备及鉴定

分别以棉铃虫组织表达的组织蛋白酶B和基因重组的大肠杆菌表达的组织蛋白酶B为抗原免疫BALB／c小鼠，选择血清抗体滴度高的免疫小鼠，取其脾细胞和Sp2／0骨髓瘤细胞进行细胞融合．经ELISA法筛选阳性克隆和有限稀释法克隆细胞，共获得分泌抗组织蛋白酶B的单克隆抗体杂交瘤细胞3株，分别命名为9D5，100E2和100H6．以ELISA法和Western blotting法对3株杂交瘤细胞株分泌的单克隆抗体进行了特异性鉴定，其中9D5和100E2能够同时识别两种不同来源的组织蛋白酶B，而100H6只能识别基因重组的大肠杆菌表达的组织蛋白酶B．     

Construction of transposon-mediated baculovirus vector and expression of green fluorescent protein in insect cells and larvae

A transposon-shuttle vector Hanpvid was constructed by using wild-type genomic DNA fromHeliothis armigera nuclear polyhedrosis virus (HaNPV). It could replicate inE. coli cells as a large plasmid and remain infectious when being induced into insect cells. Hanpvid comprises HaNPV DNA and a transposon cassette which includes a miniF replicon, a kanamycin resistance gene (kan), lacZa and an attachment site for Tn7 (attTn7). Recombinant virus rHa-FaGP was obtained after transposition of a donor plasmid carrying green fluorescent protein gene (gfp) and polyhedrin gene (ocu) into attTn7. SDS-PAGE analysis shows that both gfp and ocu genes were highly expressed inHeliothis armigera cells. Green Hemolymphocytes can be seen under a fluorescent microscope 4 d after recombinant virus rHa-FaGP infected the third-instar larvae. The infected larvae show strong green fluorescence 6 d post infection.