Objective:To construct a recombinant Efs-EmⅡ/3-Em14-3-3 vaccine against Echinococcus multilocularis (Em) using Enterococcus faecalis (Efs) as a vector, and investigate its antigenicity. Methods:The recombinant plasmid pGEX-EmⅡ/3-Em14-3-3 was transformed into Efs ATCC47077 strain using electroporation method, and the recombinant Efs-EmⅡ/3-Em14-3-3 vaccine was constructed. The plasmid was extracted for PCR amplification and identification. The recombinant Efs-EmⅡ/3-Em14-3-3 vaccine was expressed through isopropyl-β-D-thiogalactoside (IPTG) induction, and the recombinant protein was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting, and the proportion of expressed proteins in the total proteins of the bacteria was analyzed by thin layer scanning technology.Results:Using the plasmid extracted from recombinant Efs bacteria as a template, the EmⅡ/3-Em14-3-3 fusion gene with a size of about 2 554 bp could be amplified by PCR. The relative molecular mass ( Mr) of the expressed EmⅡ/3-Em14-3-3 fusion protein was approximately 119 × 10 3 by SDS-PAGE; after 5 h induction by IPTG, the expression level of target protein was high, accounting for about 9% of the total bacterial protein. Western blotting showed that the expressed protein could be recognized by mouse serum infected with alveolar hydatid cyst. Conclusion:The recombinant Efs-EmⅡ/3-Em14-3-3 vaccine is successfully constructed, and the expressed fusion protein shows specific antigenicity.

Objective:To construct a recombinant vaccine of Schistosoma japonicum (Sj) mediated by Enterococcus faecalis (Efs, rEfs-Sj26GST vaccine), and to study the expression of Sj26GST-GST fusion protein in the recombinant vaccine. Methods:The recombinant plasmid pGEX-Sj26GST was transformed into the susceptible strain Efs ATCC47077 by electroporation to construct rEfs-Sj26GST vaccine, and the plasmid was extracted for PCR identification. After induction of expression with isopropyl-beta-D-thiogalactopyranoside (IPTG), the products were analyzed and identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot.Results:After PCR identification, a 676 bp fragment was amplified, which was consistent with the length of Sj26GST amplification fragment. SDS-PAGE analysis showed that the relative molecular mass was 52 × 10 3, which was consistent with the band of Sj26GST-GST fusion protein. Western blot results showed that the Sj26GST-GST fusion protein expressed by rEfs-Sj26GST vaccine could be specifically recognized by the serum of Sj infected patients. Conclusion:The rEfs-Sj26GST vaccine is successfully constructed, and the Sj26GST-GST fusion protein expressed by recombinant vaccine can be specifically recognized by the serum of Sj infected patients.

Objective:To construct Lactococcus lactis (LL)-based recombinant LL-Eg95 (rLL-Eg95) vaccine for Echinococcus granulosus (Eg) and to examine its expression efficiency.Methods:Eg95 gene was obtained by PCR from the template of pCD-Eg95. Then, pMG36e was inserted in the Eg95 gene after double cleaving with restriction endonucleases XbaⅠ and HindⅢ to construct recombinant plasmid pMG36e-Eg95, which was transformed into E.coli BL2 (DE3) competent cells. The recombinant plasmid was extracted and identified by double restriction endonuclease digestion and was then electroporated into LL MG1363 to construct rLL-Eg95 vaccine. Then, the plamid was extracted and identified by PCR.Results:Examination of the recombinant plasmid by double restriction endonuclease digestion showed that the segment was of the expected length. PCR showed that 471 base pairs of Eg95 gene were amplified when the plasmid extracted from roxithromycin-resistant recombinant LL was used as the template. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the relative molecular mass of the Eg95 protein expressed was approximately 16.5×103 and that the amount of the expressed protein was 17% of the total bacterial proteins. Western blot findings suggested that the expressed protein could be recognized by mice serum infected with hydatid cyst.Conclusion:The rLL-Eg95 vaccine was successfully constructed, expressing Eg95 protein that has specific antigenicity.

目的 构建粪肠球菌(Efs)为载体的细粒棘球绦虫重组Efs-Eg95-EgA31疫苗,并研究其表达效率.方法 采用电穿孔技术将重组质粒pGEX-Eg95-EgA31转化粪肠球菌ATCC47077株,构建rEfs-Eg95-EgA31疫苗,抽提质粒进行PCR扩增鉴定;经IPTG诱导后进行10％SDS-PAGE和Westernblot等分析和鉴定表达产物.结果 以从rEfs抽提的质粒为模板进行PCR可扩增出约1 016 bp的Eg95-EgA31融合基因片段,SDS-PAGE显示表达产物为62.5 ku的重组蛋白,表达蛋白约占菌体总蛋白的11％;Westernblot表明重组蛋白能被细粒棘球蚴感染的鼠血清识别.结论 成功构建了细粒棘球绦虫rEfs-Eg95-EgA31疫苗,表达的融合蛋白具有特异的抗原性.

目的 研究屎肠球菌介导的铜绿假单胞菌寡肽酶B(Ef-PopB)疫苗免疫小鼠诱导的保护力及体液免疫应答反应.方法 用5×108菌落形成单位(CFU)的重组疫苗灌胃接种BALB/c小鼠,每日1次,连续3d,持续3周.首次免疫后4周用5×107 CFU的铜绿假单胞菌PA01株滴鼻攻击小鼠.攻击后两周处死小鼠,取肺计数细菌负荷,分别于免疫后0、4和6周采静脉血,分离血清,用ELISA测定IgG及其亚类和IgE水平.结果 Ef-PopB疫苗免疫组小鼠肺细菌菌落数明显低于空载体组和Ef对照组.疫苗组小鼠血清抗体IgG、IgG1、IgG2b、IgG3和IgE在初次免疫后4周均升高,在攻击后两周达较高水平.结论 铜绿假单胞菌重组Ef-PopB疫苗可诱导小鼠产生良好的保护作用和体液免疫应答.

目的 观察铜绿假单胞菌重组Ef-PopB疫苗免疫及PA01株攻击后小鼠产生的保护力、脾细胞因子和Treg的变化.方法 用5×108CFU的Ef-PopB疫苗灌胃免疫BALB/c鼠,每日接种1次,持续3d,连续3周.初次免疫后4周,取5×107 CFU的PA01株滴鼻攻击小鼠.攻击后2周杀鼠,分离肺和脾,取肺组织培养肺细菌并行菌落计数;用PaAg培养脾细胞,PCR扩增IL-2、IL-4、IFN-γ、IL-10、IL-12和Foxp3基因.结果 Ef-PopB疫苗组、空载体组和Ef对照组肺组织的菌落数分别为(0.37±0.02)×108 CFU、(7.58±0.20)×108 CFU 和(7.55±0.19)×108 CFU,差异有统计学意义(P＜0.01);从疫苗组中抽提的脾细胞基因组的DNA分别扩增出453 bp的IL-2基因、359 bp的IL-4基因、399 bp的IFN-γ基因、301 bp的IL-10基因、300 bp的IL-12基因和250 bp的Foxp3基因.结论 重组Ef-PopB疫苗可诱导小鼠脾细胞因子表达升高,产生混合型的Th1和Th2免疫应答.

目的 构建和鉴定铜绿假单胞菌重组质粒pGEX-PopB,并研究该质粒在大肠埃希菌BL21(DE3)中的表达.方法 以铜绿假单胞菌PA01标准株的DNA为模板,通过PCR扩增PopB基因.经酶切后与载体pGEX-1λT连接,构建重组质粒pGEX-PopB,电穿孔将其转化至大肠埃希菌BL21(DE3).异丙基硫代-β-D-半乳糖苷(IPTG)诱导重组菌BL21(pGEX-PopB)的表达,SDS-PAGE和Western blot对表达产物进行分析和鉴定.结果 PCR扩增出1200 bp的PopB基因;双酶切和PCR证实PopB基因成功插入pGEX-1λT中;SDS-PAGE发现重组菌表达相对分子质量约66×103的融合蛋白,其蛋白表达量约占菌体总量的20％;Western blot证实铜绿假单胞菌感染的鼠血清能特异性识别该重组蛋白.结论 成功构建了铜绿假单胞菌重组质粒pGEX-PopB,并在大肠埃希菌BL21(DE3)中高效表达具有抗原特异性的融合蛋白.

铜绿假单胞菌是一种广泛分布于自然界的条件致病菌,它是院内感染常见的病原体之一.铜绿假单胞菌对多种抗生素有耐药性,开发具有高效保护作用的疫苗是防治其感染的有效手段之一.铜绿假单胞菌钙反应蛋白Ⅴ(PcrⅤ)属III型分泌系统,具有良好的免疫原性,是疫苗开发的靶点之一.本文拟就PcrⅤ的特性、重组抗原疫苗、DNA疫苗及重组沙门氏菌疫苗等方面的研究现状进行综述.

目的 构建铜绿假单胞菌重组屎肠球菌(Enterococcus faecium,Ef)-PopB疫苗并观察其表达效率. 方法 以铜绿假单胞菌PA01株基因组DNA为模板,PCR扩增PopB基因,然后定向克隆至载体pGEX-1λT,构建重组质粒pGEX-PopB.将该质粒电穿孔转化Ef,构建rEf-PopB疫苗,抽提质粒,经双酶切和PCR鉴定后,加入异丙基硫代-β-D-半乳糖苷(IPTG)诱导其表达,采用SDS-PAGE和Western blot分别对表达产物进行分析和鉴定. 结果 PCR成功扩增出1 200 bp的PopB基因;双酶切证实PopB基因成功插入pGEX-1λT中;PCR证实rEf-PopB疫苗构建成功.SDS-PAGE检测重组疫苗表达相对分子质量约66×103的融合蛋白;Western blot检测Pa感染鼠血清能特异性识别该重组疫苗表达蛋白. 结论 成功构建了rEf-PopB疫苗,其表达产物具有免疫反应性,为Pa疫苗的研制奠定了基础.