绿脓杆菌鞭毛蛋白FlgE的重组表达及初步活性鉴定

目的 原核表达及纯化绿脓杆菌鞭毛蛋白FlgE并进行初步活性鉴定.方法 分析绿脓杆菌鞭毛蛋白FlgE的基因序列,设计带适当酶切位点的引物,用PCR方法扩增出 FlgE的编码DNA序列,与大肠杆菌表达载体pET24a同时经Nde Ⅰ/HindⅢ双酶切、纯化及连接后构建pET24a-FlgE原核表达质粒,挑选重组阳性质粒经DNA测序确认序列正确,转化至大肠杆菌BL21中进行诱导表达条件优化,使重组质粒表达目的蛋白,采用组氨酸标签亲和层析法对目的蛋白进行纯化,通过SDS-PAGE对纯化后蛋白相对分子质量进行鉴定,用试剂盒进行去内毒素化处理.在体外培养人角膜上皮细胞系细胞,加入纯化的FlgE重组蛋白,培养4h后应用实时定量PCR检测各种相关的炎性因子以反映FlgE蛋白活性.结果 可用于大肠杆菌表达系统的重组pET24a-FlgE构建成功,其编码的蛋白在BL21中获得高效表达,当诱导剂异丙基-β-D-硫代吡喃半乳糖苷浓度为1 mmol/L,在16℃、225r/min振荡培养20 h,诱导目的蛋白表达量最高,经纯化、去内毒素处理和SDS-PAGE鉴定,获得纯化的重组FlgE蛋白.角膜上皮细胞用20 μg/ml FlgE处理后,细胞内炎性相关分子IL-6和IL-8的表达量明显上升,而灭活的FlgE蛋白则丧失该刺激活性.结论 获得纯化的重组绿脓杆菌鞭毛蛋白FlgE,且可刺激角膜上皮细胞上调炎性因子IL-6、IL-8的表达.     

CD146基因重组质粒的构建及蛋白表达

目的构建CD146原核、真核表达载体,证实融合蛋白在原核细胞的诱导表达以及在胃癌细胞内的表达和定位。方法提取人黑色素瘤细胞A875的总mRNA并进行反转。以反转录的cDNA为模板PCR扩增CD146全长编码基因,分别克隆至pCDNA3.1-myc/his以及pGEX-4T-3表达载体中。原核重组质粒鉴定后转入BL21细胞中并经了诱导表达及纯化,真核表达质粒转入胃癌MKN45细胞中,分别利用westernblot和激光共焦扫描显微技术检测了重组质粒的表达以及在胃癌细胞中的定位。结果 CD146全长基因序列克隆到原核、真核表达载体中,酶切鉴定片段为1930bp。原核诱导出了GST-CD146并进行了纯化,CD146在真核细胞中表达为113KD的糖蛋白,Westernblot检测到真核转染的myc/his-CD146表达,条带约为120KD,免疫荧光显示蛋白定位在胃癌MKN细胞膜。结论成功构建了CD146原核、真核表达载体,融合蛋白在胃癌MKN45细胞表达。     

细粒棘球绦虫重组质粒pGEX-Eg95的构建及其在大肠杆菌BL21（DE3）中的表达

目的构建细粒棘球绦虫重组质粒pGEX-Eg95,并研究该质粒在大肠杆菌BL21（DE3）中的表达。方法超声粉碎细粒棘球蚴组织提取总RNA,通过RT—PCR扩增Eg95抗原编码基因;克隆至原核表达载体pGEX—1λT,构建重组质粒pGEX-Eg95;转化大肠杆菌BL21,经异丙基硫代-β—D-半乳糖苷（IPTG）诱导表达后用SDS-PAGE和Western blowing对表达产物进行分析和鉴定。结果RT-PCR扩增出471bp的Eg95抗原编码基因;双酶切证实Eg95抗原编码基因成功插入pGEX-1λT中;SDS-PAGE分析显示表达产物为相对分子质量约42500的重组蛋白,与预期结果一致,表达的蛋白约占菌体总蛋白的21％;Western blot鉴定显示重组蛋白能被细粒棘球蚴感染鼠血清识别。结论成功构建了细粒棘球绦虫重组质粒pGEX-Eg95,该质粒在大肠杆菌BL21中获得了高效融合表达,表达的融合蛋白具有特异的抗原性。     

结核分枝杆菌lhp基因原核表达载体的构建和表达

目的：构建结核分枝杆菌(MTB)lhp和基因原核表达载体并进行表达。方法：用PCR扩增MTB lhp基因，并克隆入pQE30质粒。测序正确后，再亚克隆入pET32a( )质粒，构建pQE30—CFP10和pET32a( )—CFP10重组体。结果：以重组体分别转化DH5α和BL21(DE3)菌后，经IPTG诱导，pQE30—CFP10未表达目的蛋白；而pET32a( )—CFP10则表达出Mr为20000左右重组蛋白。SDS—PAGE分析显示，IPTG诱导4h重组蛋白的表达量最高：表达蛋白以可溶性非包涵体形式存在于胞质中，表达量占全菌蛋白质的38%，用Western blot证实其具有良好的抗原性。经Ni—NTA柱纯化，获得纯度为93%的重组蛋白。结论：成功地构建原核表达载体pET32a( )—CFP10，并获得重组CFP10蛋白，为MTB重组抗原的应用奠定了基础。     

重组沙门菌表达结核分枝杆菌ESAT-6及其特异性免疫应答分析

目的 分析重组沙门菌表达的结核分枝杆菌(Mycobacterium tuberculosis,Mtb)分泌性蛋白ESAT-6诱导的特异性免疫应答.方法 将ESAT-6蛋白编码基因导入原核表达载体pYA3333中,构建重组质粒pYA33-esat.通过电穿孔法转化减毒鼠伤寒沙门菌X4550,获得重组菌X4550(33-esat).以每只105CFU剂量的重组菌滴鼻免疫C57BL/6小鼠,间隔18 d,在第2次免疫后10 d取免疫小鼠脾脏、肺脏、肠系膜淋巴结(mesenteric lymph node,MLN)及派伊尔淋巴集结(Peyer's patch,PP)细胞,以ESAT-6多肽作为刺激原,检测特异性的IFN-γ分泌细胞和IL-4分泌细胞.同时,运用CFSE方法榆测了体内抗原特异性CTL效应.结果 经沙门菌表达并运送的Mtb抗原ESAT-6能诱导特异性的免疫应答.在肺脏及PP细胞巾,检测到较高水平的IFN-γ和IL-4分泌细胞,免疫应答以Th1型为主.而在脾脏和MLN中,免疫应答呈现Th1/Th2混合应答.此外,体内CTL试验表明,重组菌能够诱导抗原特异的CTL效应,且特异性杀伤率为69.9%.结论 以滴鼻方式接种重组沙门菌,不仅能够诱导ESAT-6蛋白特异性的细胞免疫应答,还能激发特异的CTL效应,为结核病的防控提供了新的认识.     

小鼠次级淋巴组织趋化因子的原核表达及纯化

背景：次级淋巴组织趋化因子(secondary lymphoid-tissue chemokine,SLC/CCL21)是近年来发现的,具有免疫调节作用及抗肿瘤活性。 目的：构建小鼠次级淋巴组织趋化因子原核表达载体,并在大肠杆菌中高效表达重组蛋白。 方法：取C57BL/6小鼠淋巴结细胞,体外用Poly(I:C)刺激后提取RNA并反转录,以此cDNA为模板,通过PCR技术扩增出CCL21成熟蛋白编码序列。克隆入原核表达载体pBEn-SBP-SET1a,构建融合表达载体pBEn-CCL21。将表达载体转化大肠杆菌BL21(DE3),经异丙基-β-D-硫代吡喃半乳糖苷诱导后TRICINE-SDS-PAGE电泳分析和鉴定重组蛋白的表达。CCL21融合蛋白经链霉亲和柱层析纯化。 结果与结论：实验成功构建了CCL21基因的原核表达载体pBEn-CCL21,并获得相应的融合蛋白。结果显示CCL21可在大肠杆菌中高效表达,经链霉亲和柱层析纯化即可获得CCL21重组目的蛋白。     

植原体免疫主导膜蛋白A的原核表达载体构建、表达及其抗血清制备

目的 构建植原体免疫主导膜蛋白A (IdpA)的原核表达载体,表达并纯化目的蛋白,制备抗血清.方法 以重组克隆质粒pMD18-T-IdpA为模板PCR扩增IdpA基因片段,经酶切连接将IdpA基因克隆到原核表达载体pET-28a(+),重组质粒转化感受态E.coli BL21(DE3),PCR和双酶切进行鉴定.IPTG诱导重组菌表达IdpA蛋白,并进行纯化和鉴定,以纯化获得的IdpA蛋白为抗原免疫BALB/c小鼠制备抗血清,并采用ELISA和Western blot法检测抗血清的效价和特异性.结果 成功构建了原核表达载体pET-28a(+)-IdpA,并在大肠杆菌中能稳定表达IdpA蛋白,经纯化获得了纯度大于90％的高纯度目的蛋白,用纯化的IdpA蛋白免疫BALB/c小鼠,获得了效价高于1:320 000的强特异性抗IdpA蛋白抗血清.结论 成功进行了IdpA的原核表达并制备了抗血清.     

GPI修饰的ESAT-6核酸疫苗的构建及其免疫功能初探

目的构建糖基化磷脂酰肌醇(GPI)修饰的结核杆菌早期分泌性抗原靶(ESAT-6)核酸疫苗,初步分析其免疫功能。方法利用重叠PCR法构建pIRES-ESAT-6-gpi重组体,转染B16F10细胞,G418筛选阳性克隆,用RT-PCR、免疫荧光检测转染细胞的ESAT-6抗原表达情况;采集ESAT-6核酸疫苗免疫鼠血清,分别检测抗体滴度和CDC效应,免疫磁珠法分选CD4+和CD8+T细胞,CFSE/7-AAD细胞毒实验分析CTL细胞毒活性。结果测序正确的重组体pIRES-ESAT-6-gpi转染细胞后,ESAT-6表达于细胞膜表面。ESAT-6核酸疫苗免疫血清和CD8+T细胞分别通过CDC效应和细胞毒作用杀伤膜表达ESAT-6的B16F10细胞。结论构建的GPI修饰的ESAT-6核酸疫苗能够诱导免疫鼠产生体液和细胞免疫反应,杀伤膜表达ESAT-6的B16F10细胞,为进一步基于该法构建B16F10瘤苗的抗肿瘤免疫效应及机制研究奠定了基础。     

hBDNF基因原核表达重组质粒的构建及其在大肠杆菌中的表达

我们按照hBDNF基因全长编码序列设计合成引物,从人基因组DNA中扩增出760bp的片段,反向插入到pGEM-3Zf( )载体上,获得pGEMBF18克隆,限制性酶分析和DNA序列测定均证实该克隆插入片段为hBDNF基因全长编码序列。从pGEMBF18克隆中获取hBDNF全长编码片段,与原核表达载体pGEX-5T连接,构建了p5TBF34原核表达重组质粒。重组质粒转化大肠杆菌JM109,经IPTG诱导表达,SDS-PAGE特异区带分子量为43kDa,此重组蛋白占菌体可溶性蛋白总量的7.53%,Western杂交证实该特异区带具hBDNF抗原活性。     

人-鼠嵌合抗体表达载体的构建和抗人HER2抗体的表达

目的 :构建表达人 鼠嵌合抗体的通用真核表达载体 ,用于以嵌合抗体的形式表达PCR获取的小鼠抗体可变区基因 ,以便将人 鼠嵌合抗体应用于临床治疗。方法 :用人Tac抗原信号肽以及人IgCκ基因和γ1CH基因 ,构建人 鼠嵌合抗体的表达载体 ,并转染真核细胞 2 93T进行表达。用RT PCR、FACS和ELISA进行抗体表达的鉴定。结果 :利用人Tac抗原信号肽以及人IgCκ基因和γ1CH基因 ,构建了用于表达人 鼠嵌合抗体的通用真核表达载体。用本研究设计的引物 ,扩增小鼠抗人HER2抗体的V区基因片段 ,酶切后先后插入所构建的载体的相应克隆位点 ,转染 2 93T细胞可将PCR获得的小鼠抗体V区基因片段表达为人 鼠嵌合抗体。用RT PCR、FACS和ELISA证实 ,本系统可表达嵌合抗体。结论 :构建了人 鼠嵌合抗体的真核表达载体 ,并证实了其可在 2 93T细胞中表达。     

Chimeric flagellin expressed by Salmonella typhimurium induces an ESAT-6-specific Th1-type immune response and CTL effects following intranasal immunization

The flagellin component FliC of Salmonella typhimurium is capable of activating the innate immune system via specific interactions with TLR5 and can also act as a carrier of foreign antigen to elicit antigen-specific immune responses. Thus, we constructed an attenuated Salmonella strain SL5928(fliC/esat) expressing chimeric flagellin that contained the ESAT-6 antigen coding sequence of Mycobacterium tuberculosis inserted into the highly variable region of the Salmonella flagellin coding gene fliC(i). The chimeric flagellin functioned normally, as demonstrated using a flagella swarming assay and electron microscopy. To analyze the effects of chimeric flagellin, the cell-mediated immune response and cytotoxic T lymphocyte (CTL) effects specific for ESAT-6 antigen were tested after intranasal immunization of mice with flagellated Salmonella SL5928(fliC/esat). The results showed that SL5928(fliC/esat) intranasal immunization can strongly elicit an ESAT-6-specific T helper (Th) 1-type immune response in mucosal lymphoid tissues, such as nasopharynx-associated lymph nodes, lung and Peyer's patches, and a Th1/Th2 response was elicited in spleen and mesenteric lymph nodes. Furthermore, intranasal immunization of SL5928(fliC/esat) produced efficient CTL effects, as demonstrated using a 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) assay. Thus, our study revealed that Salmonella flagellin acts as a carrier for foreign antigen and triggers strong Th1 and CTL responses during intranasal immunization. Chimeric flagellin is potentially an effective strategy for the development of novel vaccines against tuberculosis in humans and animals.     

中国莱姆病螺旋体鞭毛蛋白中央区的基因克隆和表达

目的　对中国莱姆病螺旋体Borreliagarinii基因种参照菌株PD91的鞭毛蛋白中央区的编码基因进行克隆表达 ,对重组鞭毛蛋白作为莱姆病血清学诊断抗原进行初步的研究 ,并进行基因序列和氨基酸序列分析。方法　设计引物 ,用PCR技术获得PD91的鞭毛蛋白中央区的编码基因片段 ,经酶切、连接 ,插入质粒pET 30a中 ,构成重组质粒pET30a mfla,转化到大肠杆菌BL2 1,提取质粒进行酶切、DNA测序和氨基酸序列分析鉴定 ,诱导表达 ,筛选高效表达株 ,应用SDS PAGE和Westernblot鉴定重组蛋白及其抗原性。结果　成功地获得了鞭毛蛋白中央区的编码基因片段和基因重组 ,重组蛋白在宿主菌BL2 1中高效表达。Westernblot结果显示重组鞭毛蛋白的中央区与PD91的鞭毛蛋白具有相同的抗原性。经测序显示该中央区基因片段为鞭毛蛋白基因的 4 0 9～ 786bp ,与北美莱姆病螺旋体标准株B31的DNA碱基序列比较分析 ,同源性 92 %。结论　成功地对中国莱姆病螺旋体Borreliagarinii基因种的鞭毛蛋白中央区进行了克隆表达 ,并证实具有抗原性 ,为我国莱姆病血清学诊断研究提供资料。     

结核杆菌Ag85B与ESAT-6双顺反子真核表达质粒的构建及体外表达

目的构建结核杆菌Ag85B与ESAT-6双顺反子真核表达质粒。方法采用PCR的方法从结核杆菌H37Rv基因组DNA中扩增出Ag85B及ESAT-6基因,将其分别定向克隆入真核双表达载体pIRES,构建同时表达两个目的基因的双顺反子重组质粒。进行酶切分析及序列测定后,用脂质体包裹体外转染A549细胞,RT-PCR检测Ag85B及ESAT-6的表达。结果核酸序列测定证实重组质粒构建正确;该重组质粒能在体外表达Ag85B及ESAT-6mRNA。结论成功构建了结核杆菌Ag85B及ESAT-6双顺反子真核表达质粒,并在体外实现了共表达,为进一步在整体动物水平的实验研究奠定了基础。     

Restriction Fragment Length Polymorphism Analysis of Some Flagellin Genes of Salmonella enterica

Salmonellae often have the ability to express two different flagellar antigen specificities (phase 1 and phase 2). At the cell level, only one flagellar phase is expressed at a time. Two genes, fliC, encoding phase-1 flagellin, and fljB, encoding phase-2 flagellin, are alternatively expressed. Flagellin genes from 264 serovars of Salmonella enterica were amplified by two phase-specific PCR systems. Amplification products were subjected to restriction fragment length polymorphism (RFLP) analysis by using endonucleases HhaI and HphI. RFLP with HhaI and HphI yielded 64 and 42 different restriction profiles, respectively, among 329 flagellin genes coding for 26 antigens. The phase-1 gene showed 46 patterns with HhaI and 30 patterns with HphI. The phase-2 gene showed 23 patterns with HhaI and 17 patterns with HphI. When the data from both enzymes were combined, 116 patterns were obtained: 74 for fliC, 47 for fljB, and 5 shared by both genes. Of these combined patterns, 80% were specifically associated with one flagellar antigen and 20% were associated with more than one antigen. Each flagellar antigen was divided into 2 to 18 different combined patterns. In the sample of strains used, determination of the phase-1 and phase-2 flagellin gene RFLP, added to the knowledge of the O antigen, allowed identification of all diphasic serovars. Overall, the diversity uncovered by flagellin gene RFLP did not precisely match that evidenced by flagellar agglutination.     

Standardisation of polymerase chain reaction for the detection of Salmonella typhi in typhoid fever.

To improve the diagnosis of Salmonella typhi infection, a polymerase chain reaction (PCR) assay was developed for the amplification of the dH flagellin gene of S typhi. Primers were designed from dH flagellin gene sequence which will give an amplification product of 486 base pairs. In tests to study the specificity of the assay, no amplification was seen in non-salmonella strains or salmonella strains with flagellar gene other than "d". Sensitivity tests determined that 28 pg of S typhi target DNA or 3 x 10(2) target bacteria could be detected by the PCR assay. Subsequently, the PCR technique was used for detection of S typhi in blood or clot cultures from 84 patients clinically suspected of having typhoid fever, and from 20 healthy control subjects. Twenty five of 84 samples from clinically suspected cases were positive by PCR; four of which were culture negative. No amplification was seen in samples from patients who were culture positive for organisms other than S typhi or from controls. The time taken for each sample for PCR analysis was less than 48 hours compared with three to five days for blood or clot culture. PCR appeared to be a promising diagnostic test for typhoid fever.     

Detection of Salmonella typhi in the blood of patients with typhoid fever by polymerase chain reaction.

A polymerase chain reaction (PCR)-based test was developed for the detection of Salmonella typhi in the blood specimens from patients with typhoid fever. Two pairs of oligonucleotide primers were designed to amplify a 343-bp fragment of the flagellin gene of S. typhi. Amplified products were analyzed by agarose gel electrophoresis and Southern blot hybridization by using a 32P-labeled 40-base probe internal to the amplified DNA. The nested PCR with two pairs of primers could detect 10 organisms of S. typhi as determined by serial dilutions of DNA from S. typhi. The peripheral mononuclear cells from 11 of 12 patients with typhoid fever confirmed by blood culture were positive for DNA fragment of the flagellin gene of S. typhi, whereas 10 blood specimens of patients with other febrile diseases were negative. With the nested PCR, S. typhi DNAs were detected from blood specimens of four patients with suspected typhoid fever on the basis of clinical features but with negative cultures. We suggest that the PCR technique could be used as a novel diagnostic method of typhoid fever, particularly in culture-negative cases.     

Molecular characterization of a new serovar of Salmonella bongori 13,22:z39:- isolated from a lizard

Three Salmonella strains isolated from a lizard (Gallotia simoni) in the "Isla del Hierro" (Canary Islands, Spain) were serotyped as Salmonella bongori serotype 13,22:z39:-, which has not been described in the Kauffmann-White scheme of Salmonella serovars. In order to shed light on the assignment of those strains to the S. bongori species, several genes were amplified and/or sequenced. The iroB gene has been reported to be present only in S. enterica, while the invA gene has been described as being a helpful tool in distinguishing Salmonella from other bacterial species. Both genes were amplified and, as expected, only invA could be amplified. The fliC gene, encoding the phase 1 flagellin fljB gene, encoding phase 2 flagellin, and the gapA gene, which is believed to present polymorphic alleles among different subspecies, were amplified and sequenced. The sequence obtained from fliC(z39) matched with the sequences fliC(z39) obtained from other serovars. The sequence obtained from gapA clustered into the S. bongori group when it was compared to others previously described. We conclude that these three isolates are members of the S. bongori species representing a new serovar that will be described in the next supplement to the Kauffmann-White scheme.     

结核分枝杆菌cfp10-esat6融合基因原核表达载体的构建及表达

构建结核分枝杆菌cfp10-esat6融合基因及其原核表达载体,在大肠杆菌中表达融合蛋白CFP10-ESAT6。用基因拼接(GeneSOEing)法扩增cfp10-esat6融合基因,并将其定向克隆至原核表达载体pGEX-4T-1,构建原核表达重组质粒pGcfp10-esat6。重组子经限制性内切酶分析、聚合酶链式反应及测序鉴定后转化宿主菌大肠杆菌BL21,IPTG(isopropy-β-D-thiogalactoside,异丙基硫代-β-D半乳糖苷)诱导表达约42kDa带谷胱苷肽硫转移酶(Glutathione-S-TransferasesGST)蛋白标签的rCFP10-ESAT6融合蛋白,经谷胱苷肽硫转移酶融合蛋白纯化试剂盒得到纯化的融合蛋白,产物进行SDS-PAGE电泳、Western-blot鉴定。重组质粒pGcfp10-esat6中目的基因测序结果与报道序列相同;在大肠杆菌中以可溶性非包涵体形式表达;表达量约占菌体总蛋白的40%,表达蛋白纯化后获得纯度为90%左右的重组蛋白;Western印迹结果证实重组蛋白与确诊的结核病患者血清发生特异免疫反应。本研究成功构建了原核表达载体pGcfp10-esat6,获得了rCFP10-ESAT6融合蛋白,为rCFP10-ESAT6融合蛋白在结核病诊断中的应用奠定了基础。