牛流行热病毒糖蛋白G1抗原表位在毕赤酵母中的表达和抗原性鉴定

从包含牛流行热病毒G蛋白基因的质粒pMD-G中克隆G1抗原表位区基因,亚克隆进表达载体pPIC9K,构建重组载体pPIC9K-G1,线性化后电转化毕赤酵母GS115,通过G418压力和PCR法筛选阳性重组酵母进行诱导表达。经SDS-PAGE、脱糖基化分析、Western blot、ELISA、兔体免疫实验和特异性分析,表明该基因在GS115中表达并进行了适度的糖基化,表达蛋白有良好的生物学活性和特异性,可作为包被抗原,开发ELISA诊断试剂盒。     

牛流行热病毒G1抗原表位基因在大肠杆菌中的表达、纯化及抗原性鉴定

从包含牛流行热病毒G蛋白基因的质粒pMD-G中克隆G1抗原表位区基因,与表达载体pGEX-4T-1连接,成功构建重组质粒pGEX-G₁。重组质粒转化BL21（DE3）,以IPTG进行诱导,并确定了最佳表达条件的IPTG浓度为0.1mmol/L、反应温度为16℃、诱导时间为18h。可溶性表达的目的蛋白经Glutathione Sepharose TM^4B介质纯化,纯度达80%;以包涵体形式存在的重组蛋白以2%的脱氧胆酸钠洗涤、0.5%的N-十二烷基肌氨酸钠溶解、透析复性、Glutathione Sepharose TM^4B纯化后,纯度达85%以上。Western blot试验表明纯化的目的蛋白有良好的反应原性。经间接ELISA检测,测得牛流行热病毒12份阳性血清的OD490值平均为1.813±0.231,12份阴性血清的OD490值平均为0.359±0.032,差异极显著（P<0.01）。将重组蛋白作为抗原免疫兔子,试验兔均产生了高滴度的抗体,证实该蛋白有免疫原性。将目的蛋白作为包被抗原,测得8份狂犬病病毒阳性血清的OD490值平均为0.324±0.031,与所测12份阴性血清的OD490值接近,说明不存在交叉反应。以上结果均证实纯化后的重组蛋白有良好的生物学活性和特异性,可作为包被抗原,开发ELISA试剂盒。     

牛流行热病毒糖蛋白G1抗原表位在毕赤酵母中的表达和抗原性鉴定

从包含牛流行热病毒G蛋白基因的质粒pMD-G中克隆G₁抗原表位区基因,亚克隆进表达载体pPIC9K,构建重组载体pPIC9K-G₁,线性化后电转化毕赤酵母GS115,通过G418压力和PCR法筛选阳性重组酵母进行诱导表达。经SDS-PAGE、脱糖基化分析、Western blot、ELISA、兔体免疫实验和特异性分析,表明该基因在GS115中表达并进行了适度的糖基化,表达蛋白有良好的生物学活性和特异性,可作为包被抗原,开发ELISA诊断试剂盒。     

牛流行热病毒糖蛋白G1抗原表位在毕赤酵母中的表达和抗原性鉴定

从包含牛流行热病毒G蛋白基因的质粒pMD-G中克隆G₁抗原表位区基因,亚克隆进表达载体pPIC9K,构建重组载体pPIC9K-G₁,线性化后电转化毕赤酵母GS115,通过G418压力和PCR法筛选阳性重组酵母进行诱导表达。经SDS-PAGE、脱糖基化分析、Western blot、ELISA、兔体免疫实验和特异性分析,表明该基因在GS115中表达并进行了适度的糖基化,表达蛋白有良好的生物学活性和特异性,可作为包被抗原,开发ELISA诊断试剂盒。     

乙肝病毒前S1抗原突变体在大肠杆菌中的可溶性表达及鉴定

乙肝病毒前S1抗原含多个免疫优势表位及乙肝病毒肝细胞受体结合位点,具有重要的生物学功能。为使其高效、可溶性表达,在DnaStar软件辅助分析下,将前S1基因5′端复杂二级结构突变后,克隆入原核表达载体pQE-30a,转化大肠杆菌M15感受态细胞,经IPTG诱导后,获得了高水平、可溶性表达;并对其进行了纯化和鉴定,为进一步研究乙肝病毒前S1抗原的结构功能特点奠定了基础。     

SARS病毒S和N蛋白抗原表位的基因合成、融合表达及纯化鉴定

通过计算机分析SARS病毒N蛋白和S蛋白的氨基酸序列 ,初步确定含强抗原表位的N蛋白片段和S蛋白片段 ,共 5 6 0个氨基酸。选择真核和原核生物均偏爱的密码子 ,化学合成全新的SARS病毒N蛋白片段和S蛋白片段的基因序列 ,利用基因工程技术将两个基因片段串联 ,克隆至质粒Pet2 8a(+)内的NcoⅠ/EcoRⅠ位点 ,表达S蛋白片段和N蛋白片段的融合蛋白。将重组质粒转化大肠杆菌BL21(DE3) ,筛选获得了高效表达SARS病毒S蛋白片段和N蛋白片段融合蛋白的工程菌 ,表达的SARS病毒的融合蛋白约占菌体蛋白总量的 30 %左右 ,部分以可溶性形式存在。经离子交换柱和反相高压液相纯化获得了表达的融合蛋白 ,经初步鉴定 ,显示该融合蛋白有较好的抗原性和特异性.     

传染性支气管炎病毒抗原模拟表位在大肠杆菌鞭毛上的展示

目的：进一步验证通过噬菌体肽库筛选得到的2个传染性支气管炎病毒（IBV）抗原模拟表位在脱离噬菌体后仍具有与IBV抗体结合的生物学活性。方法：应用基因工程技术合成2个IBV抗原模拟表位（KSPKHSSSALHF和SIIQMNLHRPTS）的编码碱基序列,将其分别插入质粒pFliTrx,构建重组展示载体p1和p2,并分别转化大肠杆菌G1826,形成展示IBV模拟抗原表位肽的重组菌F1和F2,进行体外抗原抗体反应。结果：体外抗原抗体反应试验表明,重组菌F1与172可以与IBV阳性鸡血清特异性结合。结论：提示得到的2个抗原模拟表位在脱离噬菌体后,仍具有与IBV抗体结合的生物学活性,在研制IBV新型疫苗和诊断试剂方面具有潜在的应用价值。     

汉滩病毒核蛋白的分段表达及抗原表位分析

在大肠杆菌中对汉滩病毒Ｓ基因４种不同长度片段的重组表达质粒进行诱导表达。结果表明表达的４种ＧＳＴ－ＮＰ融合蛋白均以不溶性包含体形式存在于茵体细胞内,表达量分别占菌体蛋白总量的２９－３６％,分子量分别约为７２ｋＤ、６６ｋＤ、５４ｋＤ和４４ｋＤＤ。Ｗｅｓｔｅｒｎ ｂｌｏｔ显示５４ｋＤ和７２ｋＤ融合蛋白用酶标记汉滩病毒ＮＰＭｃＡｂｌＡ８和抗ＧＳＴ ＭｃＡｂ ３Ｃ１１染色呈阳反应。６６ｋＤ和４４ｋＤ融合蛋     

单纯疱疹病毒Ⅱ型糖蛋白G主要抗原表位的表达和抗原性检测

利用PCR拼接技术,合成含单纯疱疹病毒Ⅱ型糖蛋白G(gG2)抗原表位(氨基酸序列第561～578位)的片段,并进一步利用基因工程技术获得该表位的双拷贝片段,克隆入pET-KDO表达载体进行原核表达.经IPTG诱导后,高效表达出分子量大小约为39kDa的融合蛋白,经Western blot检测具有良好的抗原性.表达的融合蛋白经凝血酶切割和亲和层析纯化,得到双拷贝gG2(561～578aa)目的蛋白,经ELISA检测具有良好的灵敏度和特异性.该重组抗原的构建和表达可用于HSV-2特异性血清学诊断的研究.     

猪细小病毒NS1蛋白主要抗原表位区的原核表达

目的：用大肠杆菌表达猪细小病毒NS1基因的主要抗原表位区。方法：通过对GenBank发表的猪细小病毒（Porcine Par-vovirus,PPV）中国株非结构蛋白NS1的氨基酸序列分析,确定了其中抗原性较高的区域,针对此区域设计并合成了一对特异性引物,扩增出包含NS1主要抗原表位区的843bp的片段。酶切后连接到原核表达载体pET30a（＋）上,构建的原核表达载体pET30a-NS1s转化大肠杆菌BL21（DE3）感受态细胞,诱导表达。结果：重组蛋白大小约为43kD,与预期大小相符。Westernblot分析表明,该蛋白能与标准阳性血清发生特异性反应,证明该蛋白具有生物学活性。结论：NS1蛋白主要抗原表位区在大肠杆菌中成功表达,具有免疫原性。可作为鉴别诊断抗原用于PPV的临床检测。     

Expression and antigenic characterization of the epitope-G1 of the Bovine ephemeral fever virus glycoprotein in Pichia pastoris

The epitope-G₁ gene of Bovine ephemeral fever virus (BEFV) glycoprotein was synthesised by PCR and cloned into expression vector pPIC9K to construct recombinant plasmid pPIC9K-G₁. Then the pPIC9K-G₁ was linearized and transformed into Pichia pastoris GS115. The recombinant P. pastoris strains were selected by a G418 transformation screen and confirmed by PCR. After being induced with methanol, an expressed protein with 26 kDa molecular weight was obtained, which was much bigger than the predicted size (15.54 kDa). Deglycosylation analysis indicated the recombinant G₁ was glycosylated. Western blot and ELISA tests, as well as rabbit immunization and specificity experiments indicated that the target protein had both higher reaction activity and higher immunocompetence and specificity. The recombinant G₁ protein could be used as a coating antigen to develop an ELISA kit for bovine ephemeral fever diagnosis. Foundation item: National Dairy Foundation of China (2002BA518A04)     

Bovine ephemeral fever virus-induced apoptosis requires virus gene expression and activation of Fas and mitochondrial signaling pathway

Although induction of apoptosis by bovine ephemeral fever virus (BEFV) in several cell lines has been previously demonstrated by our laboratory, less information is available on the process of BEFV-induced apoptosis in terms of cellular pathways and specific proteins involved. In order to determine the step in viral life cycle at which apoptosis of infected cells is triggered, chemical and physical agents were used to block viral infection. Treatment of BHK-21 infected cells with ammonium chloride (NH₄Cl) or cells infected with UV-inactivated BEFV was seen to abrogate virus apoptosis induction, suggesting that virus uncoating and gene expression are required for the induction of apoptosis. Using soluble death receptors Fc:Fas chimera to block Fas signaling, BEFV-induced apoptosis was inhibited in cells. BEFV infection of BHK-21 cells results in the Fas-dependent activation of caspase 8 and cleavage of Bid. This initiated the dissipation of the membrane potential and the release of cytochrome c but not AIF or Smac/DIABLO from mitochondrial into cytoplasm leading to activation of caspase 9. Combined activation of the death receptor and mitochondrial pathways results in activation of the downstream effecter caspase 3 leading to cleavage of PARP. Fas-mediated BEFV-induced apoptosis could be suppressed by the overexpression of Bcl-2 or by treatment with caspase inhibitors and soluble death receptors Fc:Fas chimera. Taken together, this study provided first evidence demonstrating that BEFV-induced apoptosis requires viral gene expression and occurs through the activation of Fas and mitochondrion-mediated caspase-dependent pathways. An erratum to this article can be found at     

Expression and Antigenic Characterization of the Epitope-G1 of the Bovine Ephemeral Fever Virus Glycoprotein in Pichia pastoris

The epitope-G1 gene of Bovine ephemeral fever virus(BEFV) glycoprotein was synthesised by PCR and cloned into expression vector pPIC9K to construct recombinant plasmid pPIC9K-G1.Then the pPIC9K-G1 was linearized and transformed into Pichia pastoris GS115.The recombinant P.pastoris strains were selected by a G418 transformation screen and confirmed by PCR.After being induced with methanol,an expressed protein with 26 kDa molecular weight was obtained,which was much bigger than the predicted size(15.54 kDa).Deglycosylation analysis indicated the recombinant G1 was glycosylated.Western blot and ELISA tests,as well as rabbit immunization and specificity experiments indicated that the target protein had both higher reaction activity and higher immunocompetence and specificity.The recombinant G1 protein could be used as a coating antigen to develop an ELISA kit for bovine ephemeral fever diagnosis.     

Expression of the Streptomyces griseusα-amylase gene in Escherichia coli

Abstract The amy gene of Streptomyces griseus was not expressed in Escherichia coli cells due to the lack of recognition of the amy promoter by the E. coli RNA polymerase, as confirmed by using promoter-probe vectors. The expression of the amy gene in E. coli was detected only when the promoter-less gene was placed under the control of the lacZ promoter and was dependent on the level of IPTG added to the medium. The extracellular α-amylase detected in the culture broth seems to be released by cellular lysis. When the amy gene lacking both leader peptide and promoter was transcribed from the lacZ promoter, no α-amylase activity was detected but larger E. coli cells and inclusion bodies were observed.     

Efficient expression and characterization of hepatitis B virus preS2 antigen in Escherichia coli

The complete (encoding 55 amino acids, aa) or partial (encoding aa 1–26) preS2 region gene of hepatitis B virus (HBV) was fused to the 3-end of glutathion-S-transferase (GST) gene and expressed under the control of the inducible tac promoter in Escherichia coli at 37 °C. The fusion protein with the complete preS2 region was moderately expressed (8%) while the protein with the N-terminal 26 aa was expressed at a higher level, yielding about 20% of the total cellular proteins. The GST-preS2 (aa 1–26) protein, which contains the immunodominant epitope, was produced form the soluble protein fraction of the recombinant bacteria and purified by affinity chromatography using glutathione-agarose column. The purified preS2 fusion protein showed the antigenicity of preS2, as assessed by indirect and competitive ELISAs.     

磷脂酰丝氨酸合成酶基因pss的克隆与表达

磷脂酰丝氨酸合成酶能催化转酯反应,是定向合成特定磷脂类物质特别是磷脂酰丝氨酸的工具酶,但出发菌株产量低,很大程度上限制了酶法合成磷脂酰丝氨酸的工业化应用。利用表达载体pET-22b,实现了大肠杆菌磷脂酰丝氨酸合成酶基因在大肠杆菌BL21(DE3)中的同源高效表达。利用镍亲和柱对表达产物进行纯化,并用HPLC法对纯化后的重组酶的活力进行检测。结果表明,目的蛋白可在短时间内进行大量表达,蛋白含量是出发菌株的100倍,同时经6h的转酯反应转化率达到33%,重组磷脂酰丝氨酸合成酶活力达到69U/mg蛋白。     

HER2胞外区基因的克隆及其在大肠杆菌中的可溶性表达

采用反转录PCR和PCR方法分别克隆P185^HER2/neu胞外区基因和噬菌体M13K07g3p—N1结构域基因,然后将二偶联入pET-22b( )载体中,在大肠杆菌中进行融合表达。可溶性目的蛋白表达量占细菌可溶性表达产物总量的30％72右．并通过镍亲和层析纯化出目的蛋白。以上结果为从噬菌体抗体库中筛选抗P185^HER2/neu的抗体奠定了基础。