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     

Cloning and expression of a nitroaryl reductase gene from Streptomyces aminophilus strain MCMB411 in E. coli JM109 and Streptomyces lividans TK64

A partial genomic library was prepared in E. coli JM109 using pBR322 as vector and 2.4 kb Sau 3A I chromosomal fragment, encoding a nitroaryl reductase (nbr A) gene, from Streptomyces aminophilus strain MCMB 411. From the library, 2.4 kb fragment was recloned in E. coli JM109 and S. lividans TK64 using pUC18 and pIJ702 as vectors respectively. The recombinant plasmids pSD103 and pSD105 expressed the reductase gene and exported the enzyme in periplasmic space of E. coli and in cytoplasm of S. lividans TK64. The proteins expressed by E. coli and S. lividans had the same molecular mass (70 kD) as that expressed by parent strain, which suggested that the enzyme was processed similarly by all strains. Activities of the enzymes cloned in E. coli JM109 and S. lividans TK64 containing recombinant plasmids pSD103 and pSD105 respectively were optimum at 30 degrees C and pH 9 and requirement of cofactors was same as that of the parent strain.     

人β-actin蛋白原核表达及其单克隆抗体制备

为获得分泌抗人β-actin蛋白单克隆抗体（McAb）的杂交瘤细胞,通过在大肠杆菌中原核表达人β-actin蛋白,以纯化的人β-actin蛋白作为抗原免疫BALB/c小鼠。经过细胞的融合及筛选获得1株能稳定分泌抗人β-actin蛋白McAb的杂交瘤细胞,命名为2B4。采用间接ELISA和Western blot方法对McAb的特异性、稳定性和适用范围进行鉴定。结果显示：蛋白的相对分子质量为43 kDa,可溶于8 mol/L尿素;杂交瘤细胞上清的抗体效价为1×10^5,腹水的抗体效价为1×10^7;间接ELISA结果表明,杂交瘤细胞在体外传20代或液氮冻存3个月后,分泌的抗体效价不变;37℃保存24 h后,抗体的效价开始下降。Western blot结果显示,单克隆抗体识别人、鼠、兔和鱼的β-actin蛋白,与其发生特异性反应。2B4分泌的单克隆抗体可以广泛的应用于细胞生物学和免疫学试验,具有良好的应用价值。     

Detection of antibody to hepatitis delta virus in human serum by double antigen sandwich ELISA

A simple rapid detection of antibody to hepatitis delta virus (anti-HDV) in human serum was developed by using double antigen sandwich ELISA. HDV gene fragment encoding HDAg was isolated from a Chinese patient infected with HDV by RT-PCR, and a high-efficient expression HD-PQE31 strain was constructed with the fragment. We obtained high titer and good quality hepatitis delta virus protein purified by Ni-NTA metal-affinity chromatography, which was identified by Western blot and ELISA, then we set up the double antigen sandwich ELISA for detection of anti-HDV in human serum, and the performance of the sandwich ELISA was evaluated in terms of specificity and sensitivity. Results were: 1) The purified HDAg protein's purity was 90%, and its ELISA titer was 1/100 000. 2) 42 anti-HDV positive sera were detected and showed that the sensitivity of sandwich ELISA was higher than that of competitive ELISA (t=2.44, p<0.01). 3) The inhibitory rates for 2 anti-HDV positive sera by the specific HDAg were 74% and 93% respectively. 4) For the assay of specificity, all 60 samples infected by other hepatitis viruses and 30 normal samples were negative for anti-HDV. These results suggested that the double antigen sandwich ELISA with purified recombinant HDAg showed higher specificity and sensitivity, It can be used in routine laboratories to diagnose the HDV infection.     

COMPARISON OF THE CYTOPLASMIC AND PERIPLASMIC PRODUCTION OF RETEPLASE IN Escherichia coli

Reteplase is the recombinant type of tissue plasminogen activator variant. In this study, preplasmic and cytoplasmic (as inclusion body: IBs) production and activity of recombinant reteplase in E. coli were investigated and compared using a pET system (pET22b and pET15b). The cDNA of reteplase was cloned by polymerase chain reaction (PCR) amplification, sequenced, inserted into the vector pET 22b and pET15b, and expressed using isopropyl β-D-1-thiogalactopyranoside (IPTG). The recombinant plasmid was expressed in the form of inclusion body in pET 15b and in periplasmic space in pET22b. The obtained results of inclusion body extraction from recombinant pET22b (rpET22b) and recombinant pET15b (rpET15b) plasmids using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed a band of ～39 kD. However, the obtained results of periplasmic space extraction from rpET22b plasmid showed a very weak band, while cytoplasmic expression of reteplase (pET15b) produced a strong protein band confirmed with Western blotting. Consequently, our results demonstrated that the cytoplasmic expression system is efficient for the production of reteplase protein in prokaryote systems and a high amount of reteplase was obtained from the expressed proteins in the form of IBs. The obtained activity of rpET15b plasmid showed a higher enzyme absorbance in comparison to rpET22b plasmid. This suggests rpET15b as an appropriate candidate for reteplase production.     

人H5N1流感病毒血凝素蛋白DNA疫苗的构建及其免疫原性研究

将我国分离的首株人H5N1亚型禽流感病毒A/Anhui/1/2005作为研究对象,扩增其HA和HA1基因片段并克隆至真核表达载体pStar,构建成真核表达质粒。通过Western blot和间接免疫荧光检测方法确认,构建的重组质粒在真核细胞中成功地表达了目的蛋白HA和HA1。将重组质粒免疫BALB/c小鼠,检测免疫后外周血中HA/HA1特异性抗体的效价,并比较HA和HA1的免疫原性。结果表明,重组质粒免疫后成功地诱导了体液免疫反应,且二者的血清抗体效价无显著性差异。     

禽流感病毒H7N2血凝素HA1基因在大肠杆菌中的表达

目的　表达H7N2亚型禽流感病毒 (AIV)HA1基因 ,用于感染H7亚型禽流感病毒抗体的检测和HA1蛋白功能研究。方法　采用RT PCR方法对H7N2亚型AIVHA1基因进行扩增 ,将PCR产物克隆于pGEM T Easy载体 ,将该基因插入pGEX 4T 2中构建HA1基因原核表达载体 ,转化BL2 1大肠杆菌后 ,在IPTG诱导下表达HA1蛋白 ,Westernblot鉴定表达HA1蛋白。电洗脱方法纯化表达HA1蛋白 ,建立间接ELISA方法 ,对感染AIVH7、H9、H5亚型AIV阳性血清进行检测。结果　成功克隆H7N2亚型AIV的HA1基因 ,其核苷酸序列长度 96 6bp ,编码 32 2个氨基酸残基。构建HA1基因原核表达载体在大肠杆菌内表达出约 6 1× 10 3的HA1融合蛋白。Westernblot和ELISA方法鉴定表明 :表达HA1蛋白与感染H7亚型AIV鸡血清有反应 ,与H5、H9亚型AIV阳性血清没有反应。结论　本研究在大肠杆菌中成功表达了H7N2亚型AIVHA1基因蛋白 ,具有与感染H7亚型AIV阳性血清反应原性 ,不与H5和H9亚型AIV感染阳性血清发生反应。     

白背飞虱单克隆抗体的制备及其特性的研究

应用杂交瘤技术,制备出4株高度特异性的白背飞虱单克隆抗体,分别命名为WPH-1H9、WPH-2B6、WPH-2E12和WPH-3F12。这些抗体与其它8种昆虫未发生交叉反应,其中WPH-2B6可与白背飞虱所有虫态发生反应,其余3株只与卵和雌成虫发生反应。应用免疫双扩散法鉴定抗体类型及亚类,结果表明：WPH-2B6为IgG_2b亚类,其余均为IgG₁亚类。SDS-聚丙烯酰胺凝胶电泳和Western blot印迹分析表明,白背飞虱抗原主要由分子量分别为182、116、66.2及40 kD的4个多肽组成,其中WPH-2B6与182、116 kD的多肽结合,其余3株的单抗只与116 kD的多肽具有亲和性。最后对这些单克隆抗体在捕食作用研究中的应用潜力进行了讨论。     

产单核细胞李斯特菌actA基因在大肠杆菌中的表达及其单克隆抗体的研制

利用PCR技术从血清型1/2a的产单核细胞李斯特菌Lm-4株中扩增出actA基因,经克隆筛选和测序鉴定后,构建成该基因的原核表达载体pGEX-6P-1-actA及pET-actA,转入E·coli后,IPTG诱导目的蛋白的表达。SDS-PAGE结果表明,actA基因在两种载体中均获得表达,融合蛋白的大小分别约为120kDa和97kDa。以纯化蛋白为材料进行了ActA单抗的研制,获得4株抗ActA的单克隆抗体杂交瘤细胞株,腹水单抗ELISA效价为1∶5×104~1∶1×105。选取单抗1A5进行Westernblot分析,结果表明单抗1A5能和表达产物进行特异性反应,且与Lm-4多抗血清的Westernblot结果一致。actA基因的原核表达及单抗的研制为研究ActA蛋白的生物学活性及其致病作用奠定了基础。