刚地弓形虫DXR基因的克隆表达与生物学特性分析

目的 对刚地弓形虫1-脱氧-D-木酮糖-5-磷酸还原异构化酶(TgDXR)基因进行克隆、表达、纯化和生物学特性分析。方法 收集、纯化RH株弓形虫速殖子,提取cDNA和基因组DNA;PCR扩增TgDXR的基因片段;构建成熟TgDXR/pET-24b重组原核表达载体;经双酶切、PCR及测序鉴定阳性克隆;在大肠杆菌BL21中用IPTG诱导表达。亲和层析纯化重组TgDXR蛋白,并对该蛋白的生物学特性和酶的动力学活性进行分析。结果 从弓形虫RH株的cDNA和基因组DNA中分别扩增出长度为1 542 bp和5 464 bp的TgDXR片段,成功构建重组质粒;SDS-PAGE结果表明,目的基因在大肠杆菌中高效表达。重组蛋白的相对分子量约50 kDa。酶活性实验显示,Mg²⁺、Mn²⁺是最佳金属螯合离子,反应最佳pH值为7.5,该酶活性抑制剂膦胺霉素(Fosmidomycin)对该酶蛋白的IC₅₀为0.52 μmol/L。结论 RH株刚地弓形虫TgDXR可在原核表达系统中高效表达,该重组蛋白具有生物学活性,并有望作为弓形虫特异性药物靶标。     

查菲埃立克体重组120kDa抗原的初步应用研究

目的　克隆查菲埃立克体 12 0kDa膜表面抗原蛋白基因 ,获得纯化的重组蛋白。方法　根据查菲埃立克体(91HE17) 12 0kDa抗原蛋白基因序列设计特异性引物 ,PCR扩增查菲埃立克体 12 0kDa抗原蛋白的基因片段 ;用限制性内切酶酶切PCR扩增产物后与 pUC18载体相连接 ,经酶切和序列分析证实后 ,将目标片段定向插入原核表达载体pProEXHTB中构建pProEXHTB/ p12 0重组质粒 ;将重组质粒转化E coliDH5α ,并使转化子在IPTG的诱导下进行蛋白质表达 ;运用亲和层析和电洗脱法对重组蛋白进行纯化。结果　克隆到一大小为 10 80bp的查菲埃立克体 12 0kDa抗原蛋白的基因片段 ,用该基因与表达质粒连接 ,成功构建了重组表达质粒 ;SDS -PAGE分析显示 :在IPTG诱导下 ,重组表达质粒转化的大肠杆菌产生一分子量为 4 7kDa的目标蛋白 ,免疫印迹证明该重组蛋白能与查菲埃立克体免疫血清发生反应。用纯化的重组蛋白作免疫斑点分析 ,76份被检血清中有 2份为可疑阳性 ,但经IFA复核为阴性。结论　查菲埃立克体 12 0kDa重组抗原蛋白具有免疫反应活性 ,为以重组蛋白为基础的人单核细胞埃立克体病的血清学诊断试剂盒制备和疫苗的研制奠定了基础。     

幽门螺杆菌GGT基因的克隆及其在大肠杆菌中的表达

目的: 克隆幽门螺杆菌( H pylori)γ-谷氨酰转肽酶(γ-glutamyl transpeptidase,GGT)基因,实现GGT基因在大肠杆菌中的表达.方法: 从胃癌患者胃黏膜组织中分离培养获得H pylori,提取其基因组DNA,对GGT基因进行PCR扩增,克隆进pMD18-T载体,酶切和测序验证,构建原核表达载体pET-28a(+)-GGT,转化大肠杆菌BL21,经IPTG诱导表达重组融合蛋白,SDS-PAGE及Western blot分析检测表达产物.结果: 成功克隆了GGT基因,经酶切和测序验证正确,成功构建了pET-28a(+)-GGT质粒,高效表达出了68 kDa的融合蛋白.结论: 在大肠杆菌中成功表达了GGT重组融合蛋白,为进一步研究GGT与线粒体介导的细胞凋亡之间的关系奠定了基础.     

弓形虫蛋白激酶C受体1蛋白的表达与抗原性分析

目的构建弓形虫蛋白激酶C受体1基因的pET-30a（＋）-RACK1重组质粒,表达、纯化RACK1蛋白。方法PCR扩增RACK1基因的cDNA序列,用SacI、NcoI限制性内切酶对RACK1基因的PCR产物及pET-30a（＋）质粒进行双酶切,连接,转化大肠杆菌BL21,构建重组质粒。IPTG诱导表达,亲和层析柱纯化表达产物,SDS-PAGE和Western blotting对表达产物进行分析鉴定。结果PCR扩增出 966 bp的RACK1完整基因序列,成功构建RACK1基因的pET-30a（＋）-RACK1重组质粒,表达出约36kDa的RACK1蛋白,蛋白具有抗原性。结论成功构建弓形虫RACK1基因的pET-30a（＋）-RACK1重组质粒,纯化出RACK1蛋白,为进一步进行RACK1蛋白在弓形虫入侵分子机制中的作用研究奠定基础。     

亚洲牛带绦虫36kDa胞浆型苹果酸脱氢酶基因的表达、纯化及免疫学分析

目的对亚洲牛带绦虫胞浆型苹果酸脱氢酶基因(malate dehydrogenase,MDH)进行克隆、表达和免疫学研究。方法将亚洲牛带绦虫成虫MDH克隆到原核表达质粒pET-30a(+)中,在大肠埃希菌BL-21/DE3中用IPTG诱导表达,表达产物通过十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)进行鉴定,用镍离子金属螯合剂亲和层析柱进行纯化,纯化的重组蛋白用蛋白印迹(Western Blotting)进行免疫学分析。结果PCR、双酶切及DNA测序结果均表明pET-30a(+)-TaMDH重组质粒构建成功。SDS-PAGE结果表明目的基因在大肠埃希菌BL-21/DE3中获得高效表达,经亲和层析获得了高纯度蛋白。重组蛋白可被其免疫的SD大鼠血清识别,表明其具有免疫原性;并且能识别感染了亚洲牛带绦虫的猪血清,表明其具有免疫反应性。结论亚洲牛带绦虫苹果酸脱氢酶基因可在原核表达系统中获得具有免疫学活性的高效表达,为进一步研究该蛋白的功能奠定了基础。     

pGPU6/GFP质粒介导的表达钙网织蛋白基因的短发夹RNA的构建和鉴定

目的构建靶向钙网织蛋白(CRT)基因的短发夹RNA(shRNA)表达质粒并鉴定。方法设计并合成4对针对CRT基因的shRNA链,退火形成双链,与酶切的质粒pGPU6/GFP连接,构建4个重质粒,转化DH5а菌株,提取质粒,进行酶切鉴定和测序分析。脂质体法转染人乳腺癌MDA-MB-231细胞,通过荧光倒置显微镜判定转染效率,并通过Western印迹法检测细胞中CRT基因及蛋白的表达水平。结果经酶切鉴定筛出的重组质粒测序结果与目的序列相同,重组质粒pGPU6/GFP/Neo-CRT构建成功;转染MDA-MB-231细胞后,CRT基因在mRNA及蛋白水平的表达均降低,其中pGPU6/GFP/CRT1的抑制效果最好。结论成功构建了靶向CRT基因的shRNA表达质粒,并筛选出1种对CRT基因有显著抑制作用的shRNA。     

细粒棘球蚴(中国大陆株)线粒体苹果酸脱氢酶重组蛋白的表达、纯化及免疫特性分析

目的 对细粒棘球蚴（中国大陆株）线粒体苹果酸脱氢酶（EgmMDH）重组质粒进行原核表达、纯化，并初步鉴定重组蛋白的免疫学特性。方法 对已构建的基因工程菌株mMDH／pGEM—T／JM109进行酶切，获取目的基因。将目的基因重组到pET28a表达载体上，筛选阳性表达菌株并进行诱导表达，经SDS—PAGE鉴定重组mMDH蛋白质为包涵体后，超声破碎细胞，尿素溶解包涵体，镍柱亲和层析法纯化，获取重组蛋白。以纯化的mMDH作抗原免疫小鼠，用Westernblot和ELISA方法检测重组蛋白的免疫原性及免疫鼠血清抗体水平。结果 成功构建原核重组表达载体mMDH／pET28a／BL21，并纯化出浓度较高的重组蛋白。ELISA结果显示，重组抗原免疫小鼠诱导产生了一定水平的血清抗体；Westernblot显示，原头蚴、囊液等天然抗原能被重组抗原免疫的小鼠血清识别。结论纯化后的重组蛋白mMDH具有较强的免疫原性，有望作为包虫病候选疫苗，值得进一步深入研究。     

结核分枝杆菌阿拉伯糖基转移酶AftA的原核表达及鉴定

目的构建高效表达菌株,以大量表达结核分枝杆菌AftA蛋白,为后续的抗结核药物的筛选奠定基础。方法采用PCR法从结核分枝杆菌H37Rv基因组DNA中扩增出AftA的基因片段,将其插入原核表达质粒PQE30,构建重组质粒PQE30-aftA。将PQE30-aftA转化大肠杆菌,用IPTG诱导目的基因表达。Western-blotting免疫印迹法鉴定后纯化该表达产物。结果经核苷酸序列测定和酶切鉴定结果表明,成功构建了重组质粒PQE30-aftA。在大肠杆菌M15中用IPTG诱导表达后,获得了AftA蛋白。蛋白经SDS-PAGE分析约为69.5kD。免疫印迹分析证实目的蛋白与阳性结核病患者抗血清产生特异性反应。结论成功制备出重组的AftA蛋白,为新型抗结核药物的筛选奠定了物质基础。     

细粒棘球绦虫硫氧还蛋白过氧化物酶基因的表达及抗原性分析

目的在原核系统表达并初步鉴定细粒棘球绦虫硫氧还蛋白过氧化物酶(EgTPx)基因重组蛋白的抗原性。方法对已构建的重组质粒pMD-18T-EgTPx进行限制性酶切,获取目的基因片段后连接到表达质粒载体pET30a,构建重组表达质粒pET30a-EgTPx,转化大肠杆菌DH5α。筛选阳性克隆,经限制性酶切分析、PCR及测序鉴定后,转化大肠杆菌BL21(DE3),以异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析表达产物,对表达产物纯化后用Western-blot方法和ELISA鉴定重组EgTPx蛋白的抗原性。结果构建的重组表达质粒pET30a-EgTPx阳性克隆经PCR与双酶切鉴定,与预期结果一致,测序鉴定无基因突变,开放阅读框正确;含有pET30a-EgTPx重组表达质粒的大肠杆菌BL21(DE3)诱导后得到了高效表达,SDS-PAGE显示表达产物分子量约为27 kDa,而且主要以包涵体形式存在;Western-blot和ELISA结果表明EgTPx重组抗原可以被棘球蚴感染羊阳性血清特异性识别。结论成功构建了pET30a-EgTPx表达质粒,EgTPx重组蛋白得到了高效表达,表达产物具有良好的抗原性。     

编码弓形虫表面抗原P30基因的克隆及在E.coli中的表达

目的　构建编码弓形虫RH株表面抗原P30基因重组表达质粒 ,初步观察P30基因在E coli表达。方法　将P30基因定向克隆到分支杆菌 -大肠杆菌穿梭表达质粒热休克蛋白 70 (hsp70 )起动基因的下游的多克隆位点 ,构建重组表达质粒pBCG -P30 ;采用亚克隆技术 ,将含P30和hsp70起动基因的复合片段 ,插入表达载体 pBK -CMV质粒 ,转化大肠杆菌DH5α ,在卡那霉素阳性LB培养基平板筛选阳性重组子 ,并经双酶切及PCR扩增鉴定。重组质粒 pBK -P30转化大肠杆菌 ,IPTG诱导表达后进行SDS -PAGE和Westernboltting分析。 结果　 1)阳性重组质粒 pBCG -P30、pBK -P30经酶切和PCR鉴定 ,与预期的结果相符合。 2 )序列测定证实克隆的基因为编码P30抗原的基因。 3)P30基因在大肠杆菌诱导表达后获得4 5kDa融合蛋白 ,此抗原未被弓形虫高免兔血清识别。结论　成功构建编码弓形虫表面抗原P30重组表达质粒 ,并在大肠杆菌中获得表达 ,为弓形虫DNA疫苗的研制奠定基础     

日本血吸虫与斯氏狸殖吸虫交叉抗原的筛选和鉴定

目的筛选和鉴定可与斯氏狸殖吸虫病患者血清产生交叉反应的日本血吸虫成虫抗原或其排泄分泌（ES）抗原组分,为筛选血吸虫病特异性诊断抗原奠定基础。方法首先采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分离日本血吸虫成虫蛋白和ES蛋白,然后采用Western blot鉴定其中能够与斯氏狸殖吸虫病人血清反应的蛋白条带,最后取下对应的阳性条带进行质谱检测,对该蛋白进行分析和鉴定。结果成功采用SDS-PAGE对日本血吸虫成虫蛋白和ES蛋白进行了分离。Western blot结果显示,在ES蛋白中出现了一阳性条带,分子量约为53kDa;质谱检测结果显示该蛋白为未知蛋白。结论日本血吸虫成虫ES成分中的53kDa蛋白可能是日本血吸虫和斯氏狸殖吸虫的交叉抗原。     

Demonstration of a stimulatory protein for virus-specified DNA polymerase in phorbol ester-treated Epstein-Barr virus-carrying cells.

A heat-labile Epstein-Barr virus-specific DNA polymerase stimulatory protein having a molecular mass of 45 kDa was purified from phorbol 12-myristate 13-acetate-treated P3HR-1 cells by column chromatography. The virus DNA polymerase stimulatory protein was precipitated by sera from patients with nasopharyngeal carcinoma but not by sera from healthy donors. The interaction of the stimulatory protein with DNA polymerase was stoichiometric. Furthermore, this protein stimulated Epstein-Barr virus DNA polymerase but not herpes simplex virus type 1 or type 2 or human DNA polymerase alpha. The stimulatory protein did not alter the Km value of dTTP or DNA but did increase the Vmax of DNA polymerase. Salt concentrations between 100 mM and 150 mM KCl were optimal for this protein-induced stimulation of Epstein-Barr virus DNA polymerase activity. The presence of the stimulatory protein in the reaction mixture enhanced the sensitivity of virus DNA polymerase to phosphonoformate.     

Purification of human leukotriene C4 synthase.

Leukotriene (LT) C4 synthase, the enzyme that catalyzes the conjugation of LTA4 with reduced glutathione to form LTC4, was purified to homogeneity from the KG-1 myeloid cell line after solubilization of the microsomes utilizing a combination of 0.4% sodium deoxycholate and 0.4% Triton X-102. The solubilized enzyme was then applied to an S-hexyl-glutathione-agarose column that was eluted by the use of 7.5 mM probenecid. After removal of the probenecid by sequential concentration and dilution in an Amicon concentrator, the enzyme was additionally purified and concentrated by binding to and elution from approximately 75 mg of S-hexyl-glutathione-agarose. The enzyme was further resolved by electrophoresis with a nondenaturing Tris-glycine gel, and the LTC4 synthase activity was localized to slices 3 and 4. When the remainder of the eluate from the nondenaturing gel was precipitated by acetone and analyzed by 14% SDS/PAGE with silver staining, a single protein band of 18 kDa was associated with LTC4 synthase activity and was not present in the eluates of slices lacking activity. The overall recovery was 12.5%. In a separate preliminary purification, in which the yield was only approximately 1%, the eluates of the nondenaturing gel had also revealed a single protein of 18 kDa by SDS/PAGE, which was present only in the eluates with LTC4 synthase activity. These data identify LTC4 synthase as a protein of 18 kDa, a size consistent with its membership in the microsomal glutathione S-transferase family.     

Photoaffinity labeling of the somatostatin receptor: identification of molecular subtypes.

Pharmacological studies have suggested that the somatostatin (SS) receptor is heterogeneous and may exhibit subtypes selective for SS-14 and SS-28. Whether this heterogeneity can be explained by separate molecular forms of the receptor protein is unclear. In the present study, we have developed a novel photosensitive azido derivative of the octapeptide SS analog Tyr3 SMS (EE 581) and used it as a photoaffinity probe to characterize the molecular components of the SS receptor in five receptor positive tissues (normal rat brain, pituitary, pancreas, and adrenal cortex, and mouse AtT-20 pituitary tumor cells). [125I]EE-581 labeled specific high affinity binding sites in all these tissues (Kd range 1.3-1.67 nM). Photoaffinity labeled membrane SS receptors were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography. Three specifically labeled SS receptor proteins of 80 kilodaltons (kDa), 58 kDa, and 32 kDa were identified and exhibited a tissue-specific distribution. The 58 kDa species was the exclusive form in pancreas, adrenal cortex, and AtT-20 cells and the dominant form in brain. The 32 kDa receptor protein was expressed as a minor form (ratio of 58 kDa:32 kDa 3:1), exclusively in brain. The 80 kDa receptor was found only in the pituitary where it occurred as the sole SS receptor species. Competition experiments showed that the 58 kDa and 32 kDa receptor proteins in brain reacted with SS-14 greater than SS-28; in contrast, the 58 kDa protein in AtT-20 cells bound SS-28 greater than SS-14 suggesting the existence of distinct subtypes of the 58 kDa receptor in these two tissues. These data represent the first systematic evaluation of the molecular forms of SS receptor proteins by photoaffinity labeling in different target tissues and provide direct evidence for molecular heterogeneity and SS-14/SS-28 selectivity; a major 58 kDa protein present in most tissues, an additional 32 kDa protein uniquely expressed in brain, and an 80 kDa protein exclusive to the normal pituitary.     

Bovine brain kinesin is a microtubule-activated ATPase.

Recently, a protein called kinesin was described, which is capable of inducing movement of inert particles along microtubules. To purify this protein from bovine brain, we used the ability of kinesin to bind to taxol-stabilized microtubules in the presence of inorganic tripolyphosphate. The brain kinesin preparation contained one major polypeptide of 135 kDa and four minor polypeptides of 45-70 kDa. The minor polypeptides were eluted from a gel-permeation chromatography column at the same position as the major component. All the polypeptides of the preparation were capable of binding to the microtubules under identical conditions. The kinesin molecule is most probably a complex of these polypeptides. Brain kinesin had a very low ATPase activity (0.06-0.08 mumol X min-1 X mg-1 in 3 mM Mg2+ at pH 6.7). ATPase activity was strongly stimulated by microtubules (Vmax = 4.6 mumol per min per mg of kinesin). Microtubule-activated kinesin ATPase had a Km for ATP between 10 and 12 X 10(-6) M and a Kapp for microtubules (i.e., polymerized tubulin concentration required for a half-maximal activation) of 12-14 X 10(-6) M. Kinesin had a significant ATPase activity even without microtubules if 2 mM Ca2+ was substituted for Mg2+ (Vmax = 1.6 mumol X min-1 X mg-1; Km = 800 X 10(-6) M). Kinesin is therefore a mechanochemical ATPase that is activated by microtubules.     

日本血吸虫MBLAC1基因的克隆及生物信息学分析

目的 克隆日本血吸虫金属β内酰胺酶结构域蛋白1(Metallo-beta-lactamases domain-containing protein 1,MBLAC1)基因,并研究其编码蛋白的生物学特性。方法 以成虫虫体cDNA为模板,利用PCR技术扩增SjMBLAC1基因,并通过生物信息学技术分析该基因编码蛋白的结构特征。结果 SjMBLAC1扩增基因片段大小为711 bp,编码236个氨基酸,预测蛋白质分子量约为26 kD,理论等电点为4.84。该蛋白的第7~222位氨基酸为保守结构域,属于MBL超级家族;不存在跨膜结构域及信号肽;具有一个N-糖基化位点,在第186位氨基酸;二级结构中包含α-螺旋8.90%、β-折叠27.97%、无规则卷曲区域63.14%,推测SjMBLAC1蛋白具有9个优势B细胞抗原表位。结论 成功克隆了SjMBLAC1基因并对其编码蛋白进行了生物信息学分析,从而为开展该蛋白的生物学功能研究及筛选抗血吸虫病疫苗候选分子提供了基础。     

Direct injection and expression <Emphasis Type="Italic">in vivo</Emphasis> of full-length cDNA of the cardiac isoform of alpha-2 macroglobulin induces cardiac hypertrophy in the rat heart

Earlier studies from this laboratory have shown that the serum protein of molecular weight of 182 kDa, which plays an indispensable role in the development of cardiac hypertrophy, may be a cardiac isoform of alpha-2 macroglobulin belonging to macroglobulin family (36). Furthermore, reports on the stable expression in vivo of several reporter genes injected directly into the myocardium of rat and the approach of direct gene transfer into adult mammalian heart to characterize the activity of a cellular gene and to modulate overall cardiac function in vivo prompted us to evaluate the hypertrophy inducing potential of a full-length cDNA for the 182 kDa protein upon direct injection. The full-length cDNA of the cardiac isoform of alpha-2 M obtained from hypertrophied rat heart mRNA and cloned in an eukaryotic expression vector namely pcDNA 3.1(-) was injected directly into the rat heart; the protein whose expression was constitutively triggered by the cytomegaloviral promoter could be detected in the serum of the injected animals and it could exert its function of inducing the hypertrophy, by a highly plausible way, by which the cardiac specific 182 kDa protein may bind to many growth modulating factors in the serum, and then be targeted to cardiomyocytes by a receptor mediated mechanism (36). The development of cardiac hypertrophy was monitored by determining the heart weight-body weight ratio. This ratio along with Northern blot analysis of muscle specific marker genes such as beta-MHC, MLC-2 and ANF, and the induction of promoter activity of beta-MHC and c-fos genes monitored using chloramphenicol acetyl transferase as a reporter gene confirmed the induction of cardiac hypertrophy upon direct injection of the full-length cDNA of 182 kDa protein.