人GST-AWP1融合蛋白的原核表达及其抗体制备

为进一步研究人的一新蛋白———蛋白激酶C相关激酶 1相关蛋白 (AWP1)的结构、功能及与其相互作用的蛋白而进行GST AWP融合蛋白表达载体的构建、原核表达、纯化及其抗体的制备 .采用逆转录PCR(RT PCR)法从人ECV30 4内皮细胞中扩增AWP1cDNA编码区 ,并将其重组于谷胱甘肽硫转移酶 (GST)融合蛋白表达质粒pGEX KG中 .经酶切、序列鉴定分析后 ,用该重组质粒转化大肠杆菌BL2 1,并经异丙基 β D 硫代半乳糖苷 (IPTG)诱导产生GST AWP1融合蛋白 ,继而纯化获得了分子量约 5 6kD的融合蛋白 .将此融合蛋白免疫新西兰兔 ,经ELISA和Western印迹检测获得了效价高、免疫活性强的兔抗人多克隆抗体 .结果表明成功构建了GST AWP1融合蛋白表达载体 ,在大肠杆菌高效表达了GST AWP1融合蛋白 ,并获得高效多抗 ,为下阶段深入AWP1功能研究提供了重要的基础     

Analysis of epitope structure of PSP94 (prostate secretory protein of 94 amino acids): (I) immuno-dominant and immuno-recessive area

PSP94 is a potential biomarker for evaluating patients with prostate carcinoma. We have systematically studied the epitope structure of PSP94 by using a polyclonal antibody against human PSP94. Results of peptide mapping and ELISA tests of dose response to rabbit antiserum against human PSP94 protein showed that only the N-terminal peptides (N30 and M23) are immunoreactive while all the synthetic peptides (C28, C10) located closer to the C-terminus are completely devoid of antigenic activity with the polyclonal antibody. These results were confirmed by analysis of reciprocal competitive binding of PSP94 polyclonal antibody by the N-terminal peptides (N30 and M23) v. either recombinant GST-PSP94 fusion protein, purified recombinant PSP94, or natural PSP94 protein. To further delineate the antigenic activity of the N- and C-termini, we have also expressed N- and C-terminal half of the whole PSP94 (each 47 peptides) using the E. coli GST expression system. The recombinant N47/C47 peptides were released by thrombin cleavage from the GST fusion protein and characterized by Western blotting experiments. Dose response of the recombinant GST-PSP-N47 and -C47 peptides to PSP94 polyclonal antibody showed differential binding activities. Competitive binding of these recombinant N47/C47 proteins against the GST-PSP94 protein demonstrates that the polyclonal antibody has a higher affinity for the N47 peptide than the C47 peptide. Based on the immunological studies of both synthetic peptides and recombinant PSP94- N/C terminal proteins, we propose an epitope structure of human PSP94 with an immno-dominant N-terminus and an immuno-recessive C-terminus. J. Cell. Biochem. 65:172–185. © 1997 Wiley-Liss, Inc.     

棉铃虫核型多角体病毒orf86基因的原核表达、抗体制备与免疫印迹检测

棉铃虫核型多角体病毒(Helicoverpa armigera nucleopolyhedrovirus,HearNPV)orf86是一个功能未知的基因。从HearNPV基因组中通过PCR得到orf86基因编码序列,将其构建于原核表达载体pGEX-4T-2,转化大肠杆菌BL21获得融合表达产物。融合表达蛋白经分离纯化后免疫新西兰大白兔,制备其多克隆抗体。用ELISA测定结果表明,ORF86多克隆抗体效价为1:5.12×105。利用该抗体Western印迹检测HearNPV感染的HZAM1细胞,检测到一个36kD的目的蛋白条带,与ORF86预期大小一致。结果表明该多抗可用于对orf86编码蛋白的检测和相关功能研究。     

MAPPING OF HIV-1 GAG EPITOPES RECOGNIZED BY POLYCLONAL ANTIBODIES USING GENE-FRAGMENT PHAGE DISPLAY SYSTEM

Phage display has emerged as a powerful technique for mapping epitopes recognised by monoclonal and polyclonal antibodies. We have recently developed a simple gene-fragment phage display system and have shown its utility in mapping epitope recognised by a monoclonal antibody. In the present study, we have employed this system in mapping epitopes recognised by polyclonal antibodies raised against HIV-1 capsid protein, p24 which is derived from proteolytic cleavage of Gag polyprotein. HIV-1 gag DNA was fragmented by DNase I and the fragments (50–250 bp) were cloned into gene-fragment phage display vector to construct a library of phages displaying peptides. This phage library was used for affinity selection of phages displaying epitopes recognised by rabbit anti-p24 polyclonal antibodies. Selected phages contained sequences from two discrete regions of p24, demonstrating the presence of two antigenic regions.

The DNA sequences encoding these regions were also cloned and expressed as GST fusion proteins. The immunoreactivity of these epitopes as GST fusion proteins, or as phage-displayed peptides, was comparable in ELISA system using same anti-p24 polyclonal antibodies. The results indicate that the gene-fragment based phage display system can be used efficiently to identify epitopes recognised by polyclonal antibodies, and phage displayed epitopes can be directly employed in ELISA to detect antibodies.     

Mapping of hiv-1 Gag epitopes recognized by polyclonal antibodies using gene-fragment phage display system.

Phage display has emerged as a powerful technique for mapping epitopes recognised by monoclonal and polyclonal antibodies. We have recently developed a simple gene-fragment phage display system and have shown its utility in mapping epitope recognised by a monoclonal antibody. In the present study, we have employed this system in mapping epitopes recognised by polyclonal antibodies raised against HIV-1 capsid protein, p24 which is derived from proteolytic cleavage of Gag polyprotein. HIV-1 gag DNA was fragmented by DNase I and the fragments (50-250 bp) were cloned into gene-fragment phage display vector to construct a library of phages displaying peptides. This phage library was used for affinity selection of phages displaying epitopes recognised by rabbit anti-p24 polyclonal antibodies. Selected phages contained sequences from two discrete regions of p24, demonstrating the presence of two antigenic regions. The DNA sequences encoding these regions were also cloned and expressed as GST fusion proteins. The immunoreactivity of these epitopes as GST fusion proteins, or as phage-displayed peptides, was comparable in ELISA system using same anti-p24 polyclonal antibodies. The results indicate that the gene-fragment based phage display system can be used efficiently to identify epitopes recognised by polyclonal antibodies, and phage displayed epitopes can be directly employed in ELISA to detect antibodies.     

The expression of recombinant large myelin-associated glycoprotein cytoplasmic domain and the purification of native myelin-associated glycoprotein from rat brain and peripheral nerve

The myelin-associated glycoprotein (MAG) is a transmembrane protein of the immunoglobulin superfamily existing as two isoforms (L-MAG and S-MAG) that are differentially expressed by myelinating glial cells of the central and peripheral nervous systems, where MAG represents 1 and 0.1% of the total myelin proteins, respectively. The polypeptide chains of the two isoforms differ only by the carboxy terminus of their respective cytoplasmic domains, which most probably determine the isoform-specific functions. Here, we describe the expression of the L-MAG cytoplasmic domain as a GST fusion protein. The recombinant protein was used to raise polyclonal antibodies against the L-MAG-specific carboxy terminus and against the region of the MAG cytoplasmic domain common to both S-MAG and L-MAG. These antibodies, which function in dot blotting, Western blotting, and immunoprecipitation, were used to immunopurify native MAG from both rat brain and peripheral nerves in quantities and purity sufficient for the realization of most biochemical and functional studies. The antibodies and the recombinant and native MAG proteins provide much needed tools for the study of the common and isoform-specific properties and functions of L-MAG and S-MAG.     

hNOK抗体的制备及肺癌组织中NOK的表达检测

Novel Oncogene with Kinase-domain (NOK)是一个新的肿瘤相关基因, 其结构上?与受体蛋白相似, 具有一个蛋白激酶结构域。过量表达NOK会导致肿瘤的产生与转移。为了更进一步研究生理状态下NOK的功能, 需要制备NOK特异性的抗体。利用GST融合蛋白制备了人源NOK的多克隆抗体, 并进行了纯化。检测表明所得的抗体具有很高的滴度, 能够特异性检测NOK蛋白的表达。使用该抗体进行了人肺癌组织的免疫组化检测, 发现该抗体能够高效识别组织内源性表达的NOK蛋白, 可为肺癌的诊断提供依据。     

hNOK抗体的制备及肺癌组织中NOK的表达检测

Novel Oncogene with Kinase-domain (NOK)是一个新的肿瘤相关基因, 其结构上?与受体蛋白相似, 具有一个蛋白激酶结构域。过量表达NOK会导致肿瘤的产生与转移。为了更进一步研究生理状态下NOK的功能, 需要制备NOK特异性的抗体。利用GST融合蛋白制备了人源NOK的多克隆抗体, 并进行了纯化。检测表明所得的抗体具有很高的滴度, 能够特异性检测NOK蛋白的表达。使用该抗体进行了人肺癌组织的免疫组化检测, 发现该抗体能够高效识别组织内源性表达的NOK蛋白, 可为肺癌的诊断提供依据。     

Expression of epidermal growth factor receptor sequences as E. coli fusion proteins: applications in the study of tyrosine kinase function

To investigate the functions of key domains of the epidermal growth factor receptor (EGFR), various EGFR-derived peptide sequences were expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins. The purified fusion proteins (GST-TK0-8) were tested as substrates for the tyrosine kinase activities of the EGFR and c-src. Both the GST-TK4 fusion protein, which contains the major C-terminal tyrosine autophosphorylation sites of the EGFR, and GST-TK7, which contains the connecting sequence between the EGFR kinase domain and the C-terminal autophosphorylation domain, were strongly phosphorylated by the EGFR and c-src. Hence the candidate tyrosine phosphorylation sites present in the connecting sequences of the EGFR, as well as the known autophosphorylation sites of the EGFR, can be phosphorylated by the two tyrosine kinases. The protein GST-TK7 was phosphorylated by c-src with a KM of 5-10 microM, which indicated a potential interaction between the connecting segment of the EGFR and the c-src kinase. The GST fusion proteins were also used to map the sites recognized by two anti-EGFR monoclonal antibodies and a polyclonal serum raised against an EGFR tyrosine kinase domain fragment. The recognition site of one monoclonal antibody was determined to be in a short sequence surrounding tyr1068, a primary site of autophosphorylation in the C-terminal domain of the receptor. The anti-peptide polyclonal serum recognized only sequences in the GST-TK7 fusion protein, and hence binds to the connecting sequence between the kinase core and the C-terminal domain. These antibodies will therefore be useful reagents for studying the function of two key structural elements of the EGFR tyrosine kinase. The GST-TK fusion proteins should have many other applications in the study of EGFR catalysis and mitogenic signalling.     

GST-HAI-1融合蛋白的表达及抗人HAI-1单克隆抗体的制备

制备抗人肝细胞生长因子激活物抑制因子（HAI-1）单克隆抗体,为对HAI-1进行进一步的研究打下基础。将人HAI-1 cDNA分段克隆,构建GST-HAI-1融合蛋白原核表达载体,转化大肠杆菌后加IPTG诱导融合蛋白表达,经制备型SDS-PAGE法分离表达的GST-HAI-1融合蛋白,通过割胶、电洗脱回收融合蛋白,并以此为抗原免疫BALB/c小鼠,应用细胞融合技术制备产生抗人HAI-1单克隆抗体的杂交瘤,以ELISA、Western blot和免疫组织化学染色进行鉴定。最终获得抗人HAI-1单克隆抗体杂交瘤细胞株ZMC6,产生的单克隆抗体可特异性地与表达的GST-HAI-1融合蛋白反应,并可识别大肠组织中的膜型及脱落型HAI-1蛋白。该单克隆抗体的制备成功,为深入研究HAI-1的功能提供了有力工具。     

The E5 oncoprotein of bovine papillomavirus binds to a 16 kd cellular protein.

The E5 oncoprotein of bovine papillomavirus type 1 is the smallest known viral transforming protein. It is a 44 amino acid polypeptide asymmetrically oriented in Golgi and plasma membranes which appears to modify (either directly or indirectly) the internalization and phosphorylation of at least two growth factor receptors: EGF and CSF-1. To identify cellular proteins associated with E5, we have constructed two E5 fusion proteins, each of which contains a well-characterized epitope at the E5 amino terminus. These E5-epitope fusion proteins are biologically active, localize normally to cellular membranes and form dimers. Both monoclonal and polyclonal antibodies against the inserted epitopes specifically co-precipitate E5 and an associated 16 kd cellular protein. A transformation-defective E5 mutant containing a substitution within the hydrophobic portion of E5 is defective in its ability to bind the 16 kd protein. These findings suggest a role for E5/16 kd binding in the process of cellular transformation.     

Development of antibody-based assays for omega-conotoxin MVIIA

Omega-conotoxin MVIIA (CTX MVIIA) is a specific peptide blocker of the N-type voltage-sensitive calcium channel in neurons. The synthetic version of CTX MVIIA, Ziconotide, has been recently approved by FDA for management of severe and chronic pains. Currently, the chemical synthetic CTX MVIIA has been analyzed by RP-HPLC, and there are no chemical or immunological assays available for determination of the peptide. In this article, we report a novel method for preparation of polyclonal antibody against CTX MVIIA, and the antibody-based assays for the analysis of CTX MVIIA. The DNA sequences encoding the conotoxin were chemically synthesized and then cloned into the expression vector pGEX-2T. The GST fusion protein of CTX MVIIA was expressed in E. coli BL21 (DE3) with induction of IPTG. The purified fusion protein was used to immunize the male rabbits with standard protocols. The produced antiserum was purified through anion-exchange chromatography. Another thioredoxin (Trx) fusion protein of CTX MVIIA was employed to cross-examine the antibody against the conotoxin. Our Western blot and ELISA results show that the polyclonal antibody was capable of binding the conotoxin parts of both GST and Trx fusion proteins, and the antibody titer is 1:8192. Thus, the assays based on this antibody are useful for the conotoxin analysis.     

人早胚发育相关蛋白ZNF268的抗体制备

目的:获得纯化抗原用于制备ZNF268的多克隆抗体。方法:PCR扩增目的基因片Spacer区序列,亚克隆入融合蛋白表达载体pET28a+,构建了重组质粒pET28a+/SPA。然后将该重组质粒转化E.coliDE3(Rosseta),IPTG诱导表达,SDS-PAGE分离,获纯化融合蛋白6His-SPA。用6His-SPA免疫家兔,颈动脉取血,分离血清,从血清中获得ZNF268特异的多克隆抗体。结论:通过构建融合蛋白重组表达质粒pET28a+/SPA,用获得的初步纯化融合蛋白6His-SPA,制备了特异的ZNF268的多克隆抗体6His-SPA Ab。     

重组酶Exo的纯化及多克隆抗体制备

目的:纯化Exo重组酶融合蛋白并制备相应抗体。方法:用阴离子交换柱对蛋白进行初步纯化,然后用Ni-NTA介质填充的层析柱分离纯化含His标签的融合蛋白,用谷胱甘肽琼脂糖4B介质填充的层析柱分离纯化GST融合蛋白;二次纯化的蛋白利用硝酸纤维素膜结合法制备抗原蛋白并免疫实验动物。结果:ELISA结果显示血清抗体效价可达到1∶12 800,说明通过Western免疫印迹自制的多克隆抗体能特异地与Exo重组蛋白相互作用。结论:该蛋白纯化方法操作简单,制备的抗原纯度高,多克隆抗体特异性好。     

HIV-1辅助蛋白Vpr多肽抗体的制备与鉴定

预测Vpr蛋白的B细胞抗原表位,并利用合成的B细胞表位肽制备Vpr特异性抗体。应用生物信息学技术获得Vpr蛋白共享氨基酸序列并预测其潜在B细胞抗原表位,与载体蛋白血蓝蛋白(KLH)偶联合成多肽并免疫家兔,鉴定及纯化获得的多肽特异性抗体。软件预测显示,Vpr蛋白N端的第3～19位(N)和C端的第82～95位(C)氨基酸序列为潜在B细胞抗原表位;ELISA检测抗血清中多肽特异性抗体的效价都达到1:105以上;Western-Blotting结果显示,无论对HIV-1B亚型还是CRF07_BC重组型的Vpr蛋白,其多肽N抗体和C抗体均能特异性识别;免疫沉淀结果显示,Vpr多肽N和C抗体也能特异性结合未变性的野生型Vpr或GFP-Vpr融合蛋白。利用生物信息学技术能成功预测Vpr蛋白B细胞抗原表位,免疫所获得的抗体具有较好的特异性和应用性。     

肿瘤MUC1/Y的克隆及其胞外段在大肠杆菌中的可溶性表达、纯化和鉴定

为获得MUC1 Y全长cDNA及其胞外段蛋白 ,以用于进一步的生物学功能及肿瘤生物学治疗的研究 .利用RT PCR从HeLa细胞中扩增MUC1 Y全长cDNA ;PCR扩增其胞外段 ,克隆到原核表达载体pGEX 2T ,转化DH5a菌 ,诱导表达 ;亲和层析纯化 ;凝血酶酶切、GST活性及N端蛋白测序鉴定 ;免疫家兔制备多克隆抗体 .所得MUC1 YcDNA的开放读框为 759bp ,登录于GenBank(AF12 552 5) .其信号肽编码序列缺失 9bp ,第 3 3 1位发生G A转换 ,造成缬氨酸突变为蛋氨酸 .表达获得约 4 0kD融合蛋白GST Yex ,占菌体总蛋白 2 5%～ 3 0 % ,其中 70 %～ 80 %为可溶性 ,经亲和层析一步纯化 ,纯度 >90 % ,GST比活性为 0 2 1U μg .凝血酶酶切后的N端蛋白序列测定表明与已知序列完全一致 ,抗血清ELISA效价为 1∶2 50 0 0 0 .结果表明 ,克隆到发生碱基缺失和突变的MUC1 Y全长cDNA ,获得MUC1 Y胞外段蛋白及其多抗 ,可进一步用于相关研究 .     

单克隆非特异性抑制因子—β（MNSFβ）在大肠杆菌中的表达、抗体的制备及在小鼠子宫内膜上的组织定位

为了获得足够量的单克隆非特异性抑制因子-β蛋白（monoclonal nonspecific supressor factorβ,MNSFβ）及其抗体用于探讨MNSFβ在着床中的作用机理,本研究构建了表达质粒pBV220/MNSFβ-hCCβ,在大肠杆菌中表达了融合蛋白MNSFβ-hCCβ,用抗hCGβ抗体对表达产物进行鉴定,结果表明融合蛋白MNSFβ-hCGβ得到了正确表达,且分子量和理论值相近,表达产物MNSFβ-hCGβ经初步纯化后用于免疫Balb/C小鼠,制备抗体,同时,我们还构建了表达质粒pGEX-4T-2/MNSFβ,在大肠杆菌中表达了融合蛋白GST-MNSFβ,用融合蛋白GST-MNSFβ对融合前的免疫小鼠回忆刺激并进行检测,制备了抗MNSFβ多克隆抗体和单克隆抗体,应用所制备的多克隆抗体进行免疫组化研究,对MNSFβ在小鼠子宫内膜上进行了组织定位,结果显示,MNSFβ在着床日（受精后第4.5天）小鼠子宫非着床位点的表达和着床位点相比明显提高。     

Hsp/Hsc70相互作用蛋白CHIP在大鼠肾纤维化组织中的高表达

利用DNA重组技术 ,将编码人Hsp Hsc70相互作用蛋白CHIP(carboxylterminusofHsc 70 interactingprotein ,CHIP)全长、N端TPR及C端U box结构域的DNA片段分别构建于融合表达质粒pGEX 5X 1中 ,转化大肠杆菌 .纯化GST CHIP融合蛋白 ,制备了特异性识别人、小鼠及大鼠组织细胞内CHIP蛋白的多克隆抗体 .利用该抗体 ,对多种组织、细胞的内源性CHIP蛋白进行了检测 .发现在肾组织中 ,CHIP主要表达于皮质、髓质小管上皮细胞的胞浆中 ,且在单侧结扎输尿管所至的大鼠肾纤维化模型 (UUO)组织中 ,CHIP的表达较假手术组有显著增强趋势     

Identification of two epitopes in the carboxyterminal 15 amino acids of the E1 glycoprotein of mouse hepatitis virus A59 by using hybrid proteins.

cDNA fragments coding for the carboxy terminus of the E1 envelope glycoprotein from mouse hepatitis virus A59, a coronavirus, were cloned into the bacterial expression vector pEX. Clones expressing E1 antigenic determinants were selected with a polyclonal anti-E1 antibody and used for immunization of rabbits and for affinity purification of existing polyclonal antisera. Immunofluorescence testing and immunoperoxidase labeling of coronavirus-infected cells showed that these reagents were monospecific for E1. In addition, by using hybrid proteins containing different lengths of the E1 carboxy terminus to affinity-purify a polyclonal antiserum against E1, we have been able to define two epitopes within the last 15 amino acid residues of the protein. These epitope-specific antibodies bind to E1 in Golgi and perinuclear membranes as well as to budding viruses; they do not, however, label the plasma membrane or the membranes of post-Golgi vesicles transporting virions to the cell surface.     

Prokaryotic expression and potential application of the truncated PCV-2 capsid protein

Three pairs of specific primers were designed to amplify the F2-1, F2-2 and XF2-2 truncated sequences of ORF2 which encodes the capsid protein of porcine circovirus type 2 (PCV-2). The F2-1 sequence had most of the NLS region of ORF2, but the F2-2 and XF2-2 genes had the NLS region deleted. Truncated genes were subcloned into pET-32a(+) vectors to construct recombinant fusion expression vectors. The vectors were then transformed into Rosetta(DE3) E. coli and expressed by induction of IPTG. Expressed proteins were detected by western blotting and ELISA. The protein with best immunoreactivity was confirmed and selected, then utilized to inoculate SPF rabbits to prepare polyclonal antibodies. The protein and prepared polyclonal antibody were utilized to detect sera samples against PCV-2 from Shandong province and PCV-2 particles in PK-15 cells. In our study, three recombinant fusion proteins were successfully obtained, and the molecular weights of fusion proteins were 35.9 kDa, 33.6 kDa and 38.6 kDa respectively detected by SDS-PAGE. All of the proteins showed positive reaction with anti-PCV-2 antisera, and His-XF2-2 showed better immunoreactivity than the others. The protein of His-XF2-2 was coated as antigen in ELISA to detect the seroprevalence of PCV-2 in certain districts of Shandong province, the seropositivity rate was 27.7 % (73/264). Specific fluorescence and positive signals for PCV-2 could be detected in PK-15 cells inoculated with PCV-2 with the participation of prepared antibodies against His-XF2-2 in IFA and IPMA. Experimental results indicated that the truncated PCV-2 ORF2 gene containing most of the NLS region was successfully expressed in E. coli, and His-XF2-2 was demonstrated to have better immunoreactivity with anti-PCV-2 antisera than the other two fusion proteins. His-XF2-2 and prepared polyclonal antibodies against it had a satisfactory capability in detecting PCV-2 infection.