破骨细胞形成抑制因子TNFR结构区在大肠杆菌中表达、抗体制备及活性测定

破骨细胞形成抑制因子(OPG)对骨的重建与再吸收有重要调节作用,其TNFR结构区行使抑制破骨细胞形成与活性的功能。通过PCR将该区基因片段克隆出来,插入表达载体PET-28a质粒多克隆位点,重组质粒转入大肠杆菌BL21中进行表达,表达产物以包涵体形式存在,包涵体经变性复性后,亲合层析获得重组蛋白。纯化的产物作为抗原免疫兔,得到较高特异性的兔源多克隆抗体。利用小鼠降血钙实验检测变性复性后产物的活性,结果表明该重组蛋白有一定的生物活性。     

烟草天蛾几丁质酶的表达、纯化及多克隆抗体制备

将烟草天蛾Manduca sexta几丁质酶基因克隆入融合表达载体pET-28a,并在大肠杆菌E.coli BL21(DE3)中进行诱导表达。表达菌株经0.5 mmol/L IPTG诱导6～8 h后,几丁质酶表达并形成包涵体。在Ni²⁺-NTA亲和柱上经变、复性和纯化,得到可溶的几丁质酶。表达产物经Western 印迹鉴定。采用切胶回收的方法切割回收包涵体,并将回收产物免疫新西兰大白兔,ELISA检测抗体效价达1∶20 000。Western 印迹检测证明抗体特异性良好。     

重组人EGF-IL-18融合蛋白的表达纯化及复性

融合基因EGF-IL-18与表达载体pET32a( )连接构建融合型原核表达质粒pET32a( )-EGF-IL-18。该基因在E.coliRosetta(DE3)中获得高效表达,SDS-PAGE分析表明表达产物大部分以包涵体形式存在。以2mol/L尿素和1%TritonX-100对包涵体进行反复洗涤后,利用离子交换柱层析对包涵体进行柱上复性,结果表明离子交换层析柱上复性不仅能够获得可溶性的EGF-IL-18融合蛋白,而且产物同时得到纯化,纯度大于90%。复性的EGF-IL-18经分子筛进一步纯化后,体外实验证明具有促进人外周血单个核细胞(PBMC)IFN-g基因表达的能力。该方法简单、高效,为进一步开展EGF-IL-18的动物实验及其大量纯化制备打下基础。     

中华绒螯蟹蜕皮抑制激素基因的表达及抗体制备

蜕皮抑制激素(Moltinhibitinghormone,MIH)属于甲壳动物高血糖激素家族神经肽,对甲壳类的蜕皮起抑制作用。本研究用DNA重组技术将中华绒螯蟹(Eriocheirjaponicasinensis)的蜕皮抑制激素1(ErsMIH1)成熟肽的cDNA序列亚克隆至原核表达载体pET28a( )中,并在大肠杆菌BL21(DE3)中进行高效表达。SDSPAGE检测结果显示,融合蛋白pET-MIH1的Mr约为12kD,与理论值相符。融合蛋白的表达量约占菌体总蛋白的15%,表达产物以包涵体形式存在。对包涵体进行变性、复性及纯化处理,并以8mol/L尿素溶解的包涵体作为免疫原免疫BALB/c小鼠制备多克隆抗体。ELISA和Westernblot的结果表明制备的抗体效价高、特异性强     

原核基因工程中的包涵体

包涵体是原核基因工程的特有产物,其中表达的蛋白产物是以无活性、不溶解的形式存在。包涵体特性、蛋白回收及活性恢复是生物工程研究的重要课题。文章对包涵体特性、回收及产物提取,重组蛋白的复性及纯化作了综述。     

尖吻蝮蛇毒酸性磷脂酶A_2Ⅱ的表达及其生化特征

将尖吻蝮蛇毒酸性磷酯酶 A2 ( A.a APLA2 )基因克隆至温敏表达载体 p BLMVL2 ,在大肠杆菌 RR1中成功诱导表达 .表达产物 A.a APLA2 约占细菌蛋白质总量 2 5 % ,并以包涵体形式存在 .纯化包涵体后 ,将产物变性、复性 ,然后用 FPLC Superose TM1 2纯化 ,产物经过 SDS- PAGE检测只有单一条带 .对纯化后的表达 A.a APLA2 进行了酶活性、抑制血小板聚集活性和溶血活性的测定 .结果显示 ,A.a APLA2 的酶活性处于变性后复性江浙蝮蛇酸性磷脂酶 A2 和碱性磷脂酶A2 的酶活性之间 ,没有抑制血小板聚集活性 ,只有微弱溶血活性 .最后对 PLA2 的结构与这些活性的关系进行了讨论     

铜绿假单胞菌LexA蛋白的纯化及免疫活性分析

目的：对铜绿假单胞菌(Pseudomonas aeruginosa,PA)的LexA蛋白进行表达、纯化,并检测其免疫活性。方法： lexA基因片段插入表达载体pET32a(+),在E.coli BL21(DE3)中表达。包涵体经洗涤并用8M尿素溶解,镍离子亲合柱层析为第一步纯化,Superdex 75凝胶过滤层析作为第二步精细纯化,HPLC测定蛋白的浓度,将纯化的LexA蛋白经注射途径免疫家兔,制备兔抗LexA血清,采用免疫双扩、ELISA及Western Blot分析LexA的免疫活性。结果：LexA以包涵体形式表达,经镍离子亲合柱层析和凝胶过滤层析二步组合纯化目的蛋白,经HPLC测定目的蛋白的最终纯度为98.97%,表达及纯化的LexA具有良好的免疫活性。     

工程菌BL21-PET21a(+)-phy A表达植酸酶的研究

对来源于Aspergillusniger的植酸酶基因phyA进行了改造 ,去除了内含子和信号肽编码序列后重组到表达载体PET2 1a( + )上 ,导入大肠杆菌BL2 1 (DE3)中 ,构建工程菌株BL2 1 PET2 1a( + ) phyA。植酸酶基因在工程菌株中得到了高效表达。表达产物主要以包涵体形式存在 ,表达量达到菌体蛋白的 30 %以上。改进了包涵体复性技术 ,包涵体蛋白经复性、纯化后 ,生物活性达到 1 5 0 0U mg ,并且复性蛋白具有较好的热稳定性 ,经过 75～ 95℃、30min的热处理后 ,仍然保持了生物活性 ,同时有明显的热激活现象。热激活后最高生物活性达到 5 0 0 0U mg。这一发现对于植酸酶的应用研究具有一定的意义。     

工程菌BL21-PET21a(+)-phyA表达植酸酶的研究

对来源于Aspergillusniger的植酸酶基因phyA进行了改造 ,去除了内含子和信号肽编码序列后重组到表达载体PET2 1a( )上 ,导入大肠杆菌BL2 1 (DE3)中 ,构建工程菌株BL2 1 PET2 1a( ) phyA。植酸酶基因在工程菌株中得到了高效表达。表达产物主要以包涵体形式存在 ,表达量达到菌体蛋白的 30 %以上。改进了包涵体复性技术 ,包涵体蛋白经复性、纯化后 ,生物活性达到 1 5 0 0U mg ,并且复性蛋白具有较好的热稳定性 ,经过 75～ 95℃、30min的热处理后 ,仍然保持了生物活性 ,同时有明显的热激活现象。热激活后最高生物活性达到 5 0 0 0U mg。这一发现对于植酸酶的应用研究具有一定的意义     

羊口疮病毒蛋白ORFV035的表达、纯化和多克隆抗体的制备

克隆表达羊口疮病毒蛋白ORFV035,并制备其多克隆抗体,为后续对病毒复制、装配、形态发生和成熟过程的研究奠定基础。PCR扩增羊口疮病毒ORFV035基因,将其与质粒pET-30a(+)经Bam HⅠ和HindⅢ双酶切后连接,构建重组质粒pET30a-035。重组质粒经双酶切和测序鉴定,转化感受态大肠杆菌BL21,IPTG诱导表达,SDS-PAGE鉴定蛋白表达情况。表达产物进行超声破碎和Ni柱纯化,纯化后目的蛋白免疫小鼠,制备多克隆抗体并对其进行鉴定。成功构建了重组质粒pET30a-035,在大肠杆菌BL21中以包涵体形式高效表达。包涵体洗涤、溶解后进行Ni柱纯化,得到纯度较高的ORFV035-his融合蛋白。以纯化蛋白免疫小鼠获得多克隆抗体。Western blot检测显示该多抗可以识别天然ORFV035蛋白。成功诱导表达、纯化ORFV035蛋白并制备ORFV035多克隆抗体。     

Construction and characterization of hybrid hepatitis B antigen particles carrying a poliovirus immunogen

The hepatitis B surface antigen (HBsAg) has the unique property of assembling with cellular lipids into spherical or elongated particles of 22 nm diameter which are secreted by mammalian cells expressing HBsAg. We have studied the structural requirements for particle formation and secretion by creating in-phase insertions into different regions of the S gene of the hepatitis B virus, coding for HBsAg. Modified genes were integrated into an appropriate vector and expressed in mouse L cells. Various single and double inserts in the two major hydrophilic domains of HBsAg were compatible with particle synthesis and secretion. The level of secretion was influenced by the length of the insert, its primary structure, and the site of insertion into the HBsAg molecule. One of the inserted sequences was a synthetic DNA fragment encoding a continuous type 1 poliovirus neutralization epitope (the C3 epitope). Mammalian cells expressing the modified hepatitis B virus S gene secreted hybrid particles carrying the poliovirus antigen. The hybrid polio-HBsAg particles reacted with a monoclonal antibody specific for the C3 epitope and induced poliovirus neutralizing antibodies at low, but significant, titers in mice and at high titers in rabbits. However, the immune response to HBsAg was weaker to hybrid particles than to unmodified HBsAg particles. By cotransfection with two different plasmids carrying either modified or unmodified genes, we obtained phenotypically mixed particles containing both polio-HBsAg and HBsAg molecules. Inoculated into rabbits, the mixed particles induced high antibody titers against both poliovirus and HBsAg.     

Hepatitis B surface antigen (HBsAg) expression in plant cell culture: Kinetics of antigen accumulation in batch culture and its intracellular form

The production of edible vaccines in transgenic plants and plant cell culture may be improved through a better understanding of antigen processing and assembly. The hepatitis B surface antigen (HBsAg) was chosen for study because it undergoes substantial and complex post-translational modifications, which are necessary for its immunogenicity. This antigen was expressed in soybean (Glycine max L. Merr. cv Williams 82) and tobacco NT1 (Nicotiana tabacum L.) cell suspension cultures, and HBsAg production in batch culture was characterized. The plant-derived antigen consisted predominantly of disulfide cross-linked HBsAg protein (p24(s)) dimers, which were all membrane associated. Similar to yeast, the plant-expressed HBsAg was retained intracellularly. The maximal HBsAg titers were obtained with soybean suspension cultures (20-22 mg/L) with titers in tobacco cultures being approximately 10-fold lower. For soybean cells, electron microscopy and immunolocalization demonstrated that all the HBsAg was localized to the endoplasmic reticulum (ER) and provoked dilation and proliferation of the ER network. Sucrose gradient analysis of crude extracts showed that HBsAg had a complex size distribution uncharacteristic of the antigen's normal structure of uniform 22-nm virus-like particles. The extent of authentic epitope formation was assessed by comparing total p24(s) synthesized to that reactive by polyclonal and monoclonal immunoassays. Depending on culture age, between 40% and 100% of total p24(s) was polyclonal antibody reactive whereas between 6% and 37% was recognized by a commercial monoclonal antibody assay. Possible strategies to increase HBsAg production and improve post-translational processing are discussed.     

Process options in hepatitis B surface antigen extraction from transgenic potato

The process conditions for recombinant hepatitis B surface antigen (HBsAg) extraction from transgenic potato were examined. The effects of temperature, the reducing agent beta-mercaptoethanol (BME), and proteinase inhibitors on the level of antigenic activity of recovered HBsAg were determined. Sedimentation profiles were performed to characterize HBsAg assembly into virus-like particles. Increasing the temperature of the sample for about 1 min increased the measured HBsAg antigenic activity. The optimum temperature was around 50 degrees C. A 3-fold enhancement of the antigenic activity was obtained in extract from transgenic potato expressing HBsAg, when monoclonal antibodies were used to assay for HBsAg. When antigenic activity was determined by polyclonal antibodies, no enhancement in the antigenic activity was obtained. Temperature may affect the conformation of the a epitope to which the monoclonal antibodies bind or alter the fluidity of surface lipid regions. BME increased the antigenic activity of HBsAg up to 4-fold when monoclonal antibodies directed against the a determinant were used, but there was no increase with polyclonal antibodies. This observation suggests that BME affects the structure or presentation of the a epitope. In the presence of BME and leupeptin, a proteinase inhibitor, higher antigenic activity was obtained. Leupeptin might protect the antigen, which might become more susceptible to proteolytic degradation after reduction, as a result of stimulation of sulfhydryl proteases. Although both temperature and BME increased the antigenic activity of HBsAg individually, when combined their interaction was antagonistic, resulting in reduced antigenic activity. Different proteinase inhibitors, including leupeptin, aprotinin, E-64, pefabloc, and pepstatin, had no significant effect on HBsAg from potato extract in a 2 h period in the absence of BME. The sedimentation profile of potato-produced HBsAg was determined in 5-30% sucrose gradients. Yeast-derived recombinant HBsAg was used as a positive control. The HBsAg from transgenic potato showed sedimentation and density properties that are very similar to the yeast-produced antigen, indicating assembly into virus-like particles. BME treatment did not change the sedimentation profile.     

Epitopes associated with a synthetic hepatitis B surface antigen peptide

A synthetic peptide (SP1), corresponding to the amino acid residues 122 through 137 of the major polypeptide derived from hepatitis B surface antigen (HBsAg), subtype ayw, was analyzed for the presence of the major epitopes of HBsAg. Both a cyclic form, produced by introduction of an intrachain disulfide bond, and a linear form of the peptide were characterized. A panel of monoclonal antibodies with defined specificity for the cross-reactive group a antigenic determinant(s) and for the y and w subtype specificities was used for this analysis. The cyclic, but not the linear, form of SP1 reacted with five of 14 anti-a monoclonal antibodies, demonstrating that the cyclic peptide contains a conformation-dependent a epitope. Only one anti-a antibody was found to react with both cyclic and linear forms of SP1. Because SP1 failed to react with the remaining 8 anti-a monoclonal antibodies, it was concluded that the a antigenic reactivity associated with HBsAg contains an additional epitope(s) unrelated to that expressed on SP1. Both cyclic and linear SP1 reacted with three of three anti-y monoclonal antibodies, indicating that a sequential y epitope is also present on SP1; no w reactivity was detected. Analysis of the idiotypes associated with the monoclonal antibodies showed those that combined with cyclic SP1 also inhibited the binding of a common human anti-HBs (CHBs) idiotype with its rabbit anti-idiotype serum, whereas a monoclonal antibody that did not react with the cyclic SP1 epitope failed to inhibit the CHBs idiotype-anti-idiotype reaction. Thus, the conformational a epitope present on cyclic SP1 appears to contain the predominant epitope recognized by humans in response to a natural HBV infection.     

Generation and Characterization of High Affinity Humanized Fab Against Hepatitis B Surface Antigen

5S is a mouse monoclonal IgG1 that binds to the ‘a’ epitope of the Hepatitis B surface antigen (HBsAg) and tested positive in an in vitro test for virus neutralization. We have earlier reported the generation of humanized single chain variable fragment (scFv) from the same. In this article we report the generation of a recombinant Fab molecule by fusing humanized variable domains of 5S with the constant domains of human IgG1. The humanized Fab expressed in E. coli and subsequently purified, retained a high binding affinity (K_D = 3.63 nmol/L) to HBsAg and bound to the same epitope of HBsAg as the parent molecule. The humanized Fab also maintained antigen binding in the presence of various destabilizing agents like 3 M NaCl, 30% DMSO, 8 M urea, and extreme pH. This high affinity humanized Fab provides a basis for the development of therapeutic molecules that can be safely utilized for the prophylaxis and treatment for Hepatitis B infection.     

一种联合检测乙型肝炎病毒前S1抗原与核心抗原方法的建立及其与病毒核酸检测结果的一致性

本研究以与血清中HBV DNA含量高度相关的两种HBV抗原(前S1抗原与核心抗原)为靶标,建立了联合检测这两种HBV核酸相关抗原(NRAg)的双抗体夹心法ELISA试剂.对系列稀释血清的检测表明,该试剂的平均分析灵敏度为103.2基因组拷贝/mL(95%可信限102.2-4.2基因组拷贝/mL),显著高于前S1抗原或核心抗原的单独检测.对994份HBsAg阴性血清的检测结果表明NRAg ELISA的特异性为99.7%(95%可信限:99.1%～99.9%).对271份临床慢性肝炎血清进行检测,结果NRAg ELISA与HBV DNA结果的总符合率达96.3%(95%可信限:93.3%～98.2%),NRAg ELISA的读值/临界值比(S/CO)与HBV基因组拷贝数呈正相关.利用NRAg试剂,发现了1例HBsAg"a"抗原表位突变的变异株.这些结果显示HBV NRAg ELISA与HBV DNA具有高度相关性,并能够检测出HBsAg抗原变异株,有望成为HBsAg变异株筛选的有力工具,并为广大基层医疗单位提供一种便捷的替代HBV DNA定性检测的手段.     

Genetically engineered diphtheria toxin fusion proteins carrying the hepatitis B surface antigen

Tripartite fusion proteins comprising the nontoxic mutant protein CRM228 of diphtheria toxin (DT), the hepatitis B virus surface antigen (HBsAg), and beta-galactosidase were obtained by expression of hybrid genes from the pR promoter of bacteriophage lambda and purification by affinity chromatography. The antigenicity and immunogenicity of the individual protein constituents were analyzed. A major neutralizing epitope of DT was inactivated by the HBsAg insertion into the DT B fragment. The fusion proteins elicited antibodies reactive with 22 nm HBsAg particles. This suggests a novel approach towards the use of DT mutants as immunogenic carriers of heterologous antigens.     

杜氏盐藻外源基因稳定表达系统的构建

建立了一种单细胞海水绿藻--杜氏盐藻(Dunaliella salina Teod.)的外源基因稳定表达系统.通过电激法将携带乙肝病毒表面抗原基因(HBsAg)和氯霉素乙酰转移酶基因(CAT)的质粒转入盐藻细胞内,CAT基因为筛选基因.PCR和Southern杂交结果显示,HBsAg基因已经整合到盐藻基因组中.Northern杂交结果表明,转化成功细胞内的该基因已转录成mRNA.HBsAgELISA和Western杂交检测证明,HBsAg蛋白在转化的盐藻细胞内稳定地表达.同时,PCR和Southern杂交显示,CAT基因也已整合到盐藻基因组中.且CATELISA检测证明,CAT蛋白在转化体中也已稳定地表达.进一步对转化盐藻进行无氯霉素筛选培养,60代后,HBsAg基因依然稳定地存在并表达.本实验第一次报道了外源基因在杜氏盐藻细胞内的稳定表达.     

一种新的乙型肝炎病毒表面抗原结合蛋白的筛选、表达及其生物学活性的初步研究

为了研究乙肝病毒侵染肝细胞过程中的功能蛋白 ,通过印迹免疫分析技术从人肝cDNA噬菌体表达库中筛选出一株编码乙肝表面抗原结合蛋白 (hepatitisBsurfaceantigenbindingprotein ,HBsAg BP)的cDNA克隆 .基因测序结果表明 ,该cDNA具有独立的开放阅读框架 ,编码 1个由 344个氨基酸残基构成的可溶性蛋白分子 ,属于免疫球蛋白超家族成员 .将该基因克隆到原核表达载体pTriplEx后 ,在E .coliXL1 Blue菌株中获得 4 4kD的重组蛋白 .重组蛋白经Western印迹和ELISA实验证明具有与乙肝表面抗原特异性结合的能力 .进一步经流式细胞仪实验显示 ,在纯化的重组蛋白存在的情况下 ,天然的HBsAg与肝细胞株HepG2的亲和力显著增高 .结果显示 ,该乙肝表面抗原结合蛋白可能是介导乙肝病毒对肝细胞亲和侵染的可溶性辅助受体 .     

杜氏盐藻外源基因稳定表达系统的构建(英文)

A stable transformation system for the expression of foreign genes in the unicellular greenmarine alga (Dunaliella salina Teod.) was established. Using electroporation, the alga was transformed witha plasmid containing the hepatitis B surface antigen (HBsAg） gene and the chloramphenicol acetyltransferase(CAT) gene as a selectable gene. PCR and Southern blotting analysis indicated that the HBsAEgene wasintegrated into the D. salina genome. Northern dotting analysis showed that the HBsAg gene was expressedat the mRNA level. The stable expression of HBsAg protein in transformants was confirmed by HBsAgenzyme-linked immunosorbent assay (HBsAg EUSA) and Western blotting analysis. Also, PCR and Southernblotting analyses showed that the CA Tgene was integrated into the D, salina genome, and CAT EUSAindicated that CAT protein was stably expressed in the cells. The introduced HBsAg DNA and HBsAgprotein expression were stably maintained for at least 60 generations in media devoid of chloramphenicol.This is the first report of the stable expression of foreign genes in D. salina.