日本血吸虫26ku谷胱甘肽硫—转移酶基因的克隆及分析

目的 扩增日本血吸虫２６ｋｕ谷胱甘肽硫－转移酶（Ｓｊ２６ＧＳＴ）基因片段,构建其重组原核载体,以研制ＳｊＧＳＴ重组克隆。方法 根据已知基因序列设计一对引物,运用ＲＴ－ＰＣＲ技术扩增Ｓｊ２６ＧＳＴ目的基因ｃＤＮＡ片段,将其克隆到ｐＢｌｕｅｓｃｒｉｐｔ（ＫＳ）质粒中,并经酶切、ＰＣＲ和测序鉴定。结果 获得ＧＳＴ－ｐＢｌｕｅｓｃｒｉｐｔ（ＫＳ）重组克隆。结论 本实验获得Ｓｊ２６ＧＳＴ重组克隆,为进一步研     

日本血吸虫重组BCG—Sj26GST疫苗对小鼠免疫应答的影响

采用日本血吸虫重组ＢＣＧ－Ｓｊ２６ＧＳＴ疫苗免疫ＢＡＬＢ／ｃ小鼠。免疫８周时,断头取血,取脾脏,取腹腔巨噬细胞。以淋巴细胞刺激指数（ＳＩ）反映细胞增殖能力,以ＮＯ湫得检测巨噬细胞吞噬活性,以ＥＬＩＳＡ试剂盒检测血清及脾淋巴细胞培养上清的白细胞介素２（ＩＬ－２＿和干扰素γ（ＩＦＮ－γ）的含量。结果表明,ｒＢＣＧ－Ｓｊ２６ＧＳＴ疫苗以１０＾－６ＣＦＵ剂量经皮下免疫小鼠后,小鼠脾淋巴细胞增殖能力明显高于     

日本血吸虫26ku抗原基因在分枝杆菌和大肠杆菌中的高效表达

构建了大肠杆菌－分枝杆菌穿梭质粒ｐＢＣＧ－２０００，并将日本血吸虫２６ｋｕ抗原（Ｓｊ２６ＧＳＴ）基因在分枝杆菌中进行了克隆和表达。以质粒ｐＢｌｕｅｓｃｒｉｐｔ ｓｋ为骨架，分别克卫生玫分枝杆菌质粒复制基因、Ｎｅｏ基因（编码卡那霉素抗性）以及人结核杆菌热休克蛋白７０（ｈｅａｔ ｓｈｏｃｋ ｐｒｏｔｅｉｎ，ｈｓｐ７０）的启动子，并带有质粒ｐＢｌｕｅｓｃｒｉｐｔ的多克隆位点，构建成分枝杆菌－大肠杆菌穿梭     

日本血吸虫重组BCG-Sj26GST疫苗对小鼠免疫应答的影响

采用日本血吸虫重组ＢＣＧ－Ｓｊ２６ＧＳＴ疫苗免疫ＢＡＬＢ／ｃ小鼠。免疫８周时,断头取血,取脾脏,取腹腔巨噬细胞。以淋巴细胞刺激指数（ＳＩ）反映细胞增殖能力,以ＮＯ释放量检测巨噬细胞吞噬活性,以ＥＬＩＳＡ试剂盒检测血清及脾淋巴细胞培养上清的白细胞介素２（ＩＬ－２）和干扰素－γ（ＩＦＮ－γ）的含量。结果表明,ｒＢＣＧ－Ｓｊ２６ＧＳＴ疫苗以 １０~６ＣＦＵ剂量经皮下免疫小鼠后,小鼠脾淋巴细胞增殖能力明显高于对照组、载体组和ＢＣＧ组（均为Ｐ＜０．０５）;小鼠腹腔巨噬细胞ＮＯ的含量明显高于对照组和载体组（均为Ｐ＜０．０１）;小鼠血清ＩＬ－２的含量明显高于对照组（Ｐ＜０．０１）和载体组（Ｐ＜０．０５）;而小鼠脾淋巴细胞培养上清ＩＬ－２的含量分别高于对照组４４％、载体组４８％和ＢＣＢ组４２％;小鼠血清ＩＦＮ－γ的含量分别高于对照组２０％、载体组１９％和ＢＣＧ组１３％,均有升高趋势、结果提示,ｒＢＣＧ－Ｓｊ２６ＧＳＴ疫苗能明显增强小鼠的免疫应答反应,其抗感染作用一方面与ＢＣＧ本身的佐剂特性有关,另一方面与机体产生抗Ｓｊ２６－ＧＳＴ特异性抗体有关。     

血吸虫重组BCG—Sj26GST疫苗接种后保护力的观察

为了探讨血吸虫重组ＢＣＧ－Ｓｊ２６ＧＳＴ疫苗免疫鼠后对尾蚴攻击感染的保护性作用，采用１０＾６ＣＦＵ和１０＾６ＣＦＵ重组ＢＣＧ－Ｓｊ２６ＧＳＴ疫苗皮下接种ＢＡＬＢ／Ｃ鼠，接种后８周用日本血吸虫尾蚴进行攻击感染，感染后６周剖杀小鼠，计算减虫率、减卵率，同时进行虫卵肉芽肿周长和面积测量，以及抗体水平和抗原水平测定，同时设有ＰＢＳ对照组。结果表明重组ＢＣＧＴ－Ｓｊ２６ＧＳＴ疫苗免疫小鼠后，１０＾６ＣＦＵ、     

血吸虫重组BCG-Sj26GST疫苗接种后保护力的观察

为了探讨血吸虫重组ＢＣＧ－Ｓｊ２６ＧＳＴ疫苗免疫鼠后对尾蚴攻击感染的保护性作用,采用１０６ＣＦＵ和１０８ＣＦＵ重组ＢＣＧ－Ｓｊ２６ＧＳＴ疫苗皮下接种ＢＡＬＢ／Ｃ鼠,接种后８周用日本血吸虫尾蚴进行攻击感染,感染后６周剖杀小鼠,计算减虫率、减卵率,同时进行虫卵肉芽肿周长和面积测量,以及抗体水平和抗原水平测定,同时设有ＰＢＳ对照组。结果表明重组ＢＣＧ－Ｓｊ２６ＧＳＴ疫苗免疫小鼠后,１０６ＣＦＵ、１０８ＣＦＵ组与对照组相比,减虫率分别为２７．３４％、１６．６４％,减卵率分别为５５．７５％、６０．５０％,虫卵肉芽肿的平均周长和平均面积、免疫后血清抗体水平差别均具有非常显著意义;１０８ＣＦＵ与１０６ＣＦＵ组相比,血清抗原水平差别具有显著意义,说明血吸虫重组ＢＣＧ－Ｓｊ２６ＧＳＴ疫苗是一种比较理想的新型疫苗,值得进一步深入研究     

中山医科大学博士后吴忠道发现新的日本血吸虫基因

中山医科大学基础医学博士后吴忠道在站研究发现新的日本血吸虫基因，已被ＧｅｎＢａｎＫ接受进入新序列号。吴博士在站２年间由余新炳教授指导下开展研究工作。通过对已构建的日本血吸虫（中国大陆株）成虫ｃＤＮＡ文库（ＳｊｃＤＮＡ）的免疫学筛选分离和鉴定血吸虫重组抗原ｃＤＮＡ克隆，应用对再感染具有抵抗力的个体混合血清，从ＳｊｃＤＮＡ文库中筛选到５个日本血吸虫抗原基因（ｃＤＮＡ）克隆，１个为ＳｊＧＳＴ基因克隆，另４个为日本血吸虫未知基因重组克隆，其ＥＳＴ序列均已被ＧｅｎＢａｎＫ接受并获得进入新序列号，经初步的免…     

日本血吸虫谷胱甘肽转移酶基因在真核表达载体中的亚克隆

目的 将日本血吸虫谷胱甘肽转移酶（ＧＳＴ）基因片段克隆到真核表达载体ＰＢＫＣＭＶＫ ,以便以进行酸疫苗的研究。方法 特定核苷酸引物的设计和合成,ＴＲＩＺＯＬ试剂以日本血吸虫成虫ＲＮＡ〈ＲＴ－ＰＣＲ法扩增ＧＳＴ基因编码序列,将扩增产物连接ＰＧＥＭ－Ｔ克降体,再亚克隆到真核表达载体ＰＢＫＣＭＶ中。结果 ＲＴ－ＰＣＲ法特异性扩增出ＳｊＧＳＴ编码基因片段。其大小约为６７０ｂｐ,经双酶切、ＰＣＲ鉴定表明所构     

IL—12对日本血吸虫重组SjGST—Sj32免疫小鼠攻击感染后虫 …

目的 观察白细胞介素－１２（ＩＬ－１２）对重组日本血吸虫谷胱苷肽－Ｓ－转移酶－３２ｋＤ融合蛋白（ｒＳｊＧＳＴ－Ｓｊ３２）蛋白免疫小鼠肝脏虫卵肉芽肿大小的影响。方法 ＢＡＬＢ／ｃ小鼠３６只随机分别ＩＬ－１２加ｒＳｊＧＳＴ－Ｓｊ３２组、ｒＳｊＧＳＴ－Ｓｊ３２组和磷酸盐缓冲液（ＰＢＳ）组，第３次免疫后４周，经腹部贴片感染１０条尾蚴，４５天后杀鼠取肝组织，常规染色，光镜下进行组织学观察及单个虫卵肉芽肿大小     

日本血吸虫大陆株26KDa基因重组蛋白保护性免疫力的研究

本文报道了ｒＳｊｃ２６ＧＳＴ分别抗原可诱导宿主产生抗日本血吸虫感染的保护性免疫力，获得免疫的小鼠虫贡荷与肝、脾组织中虫卵负荷均下降，减虫率与肝、脾减卵分别为３０与５７．１１％、７９．９７％。ＥＬＩＳＡ测免疫鼠血清特异性抗体水平明显升高。提示ｒＳｊｃ２６ＧＳＴ作为日本血吸虫病候选疫苗矍有广泛应用前景。     

Expression of Foreign Gene in Mycobacterium Regulated by Human Mycobacterium Tuberculosis Heat Shock Protein 70 Promoter

CHENGJizhong,male,bornin1968,LecturerThisprojectwassupportedbyPrimaryFoundationofChina(No.94-Y-19),ChineseNationalNaturalScienceFoundation(No.39480022),andChineseHealthMinistryFoundation(No.94-1-144).Heatsh0ckproteinrepresentsahighlyconservedgr0up0fproteinsinb0thprokary0ticandeukaryoticcelltypeswhichisstronglyupregulatedinresponsetoava-rietyofdifferentstressconditi0ns,suchasheat,irradiation,andthepresenceofoxida-tiveandt0xicagents-Theyp1ayanimpor-tantroleintrans-membranetransferandcor…     

The construction ofSchistosoma Japonicum vaccine BCG-Sj26GST and its identification

Summary The expression of foreign gene,Schistosoma Japonicum 26 ku antigen (Sj26GST),in Bacillus Calmette-Guerin (BCG),Mycobacterium CM. smegmatis) andEscherichia coli (E. coli) were studied. The cDNA fragment encoding Sj26GST was amplified by PCR using plasmid pGEX, which could express Sj26GST inE. coli as template. The Sj26GST cDNA was cloned into the downstream of humanM. tuberculosis heat shock protein (hsp) 70 promoter with correct reading frame, and then the DNA fragment containing hsp70 promoter and Sj26GST gene were subcloned together intoE. coli-Mycobacteria shuttle plasmid pBCG-2000 to construct the expression shuttle plasmid pBCG-Sj26. The recombinant BCG andM. smegmatis mc²155, which were electroplated with pBCG-Sj26, could express Sj26GST and the recombinantSchistosoma Japonicum vaccine BCG-Sj26GST was made. The recombinant Sj26GST (rSj26GST) were soluble and could be observed on SDS-PAGE at molecular weight of 26 ku. The content of rSj26GST accounted for 15% and 10% of total bacterial protein in BCG andM. smegmatis respectively. The results of Western blot showed the combination of rSj26GST with antibody of GST. This project was supported by Primary Foundation of China (No. 94-Y-19), Chinese National Nature Science Foundation (No. 339480022) and Chinese Health Foundation (No. 94-1-131).     

The Construction of Schistosoma Japonicum Vaccine BCG Sj26GST and Its Identification

Ｖａｃｃｉｎｅｓａｒｅｔｈｅｍｏｓｔｅｃｏｎｏｍｉｃａｎｄｅｆ－ｆｅｃｔｉｖｅｔｏｏｌｓｉｎｐｒｅｖｅｎｔｉｏｎｏｆｄｉｓｅａｓｅｓ．ＢＣＧｈａｓｆｏｌｌｏｗｉｎｇｕｎｉｑｕｅｆｅａｔｕｒｅｓ：Ｔｈｅｐｒｏｄｕｃｔｓｃａｎｂｅｕｓｅｄｉｎｉｍｍｕｎｉｔｙｐｒｏｔｅｃｔｉｖｅｅｘｐｅｒｉ－ｍｅｎｔｄｉｒｅｃｔｌｙｗｉｔｈｏｕｔｐｕｒｉｆｉｃａｔｉｏｎａｎｄｐｒｏ－ｃｅｄｕｒｅｉｓｓｉｍｐｌｅａｎｄｃｈｅａｐ;ｓｉｍｐｌｅｏｎｅｖａｃ－ｃｉｎａｔｉｏｎｃａｎｉｎｄｕｃｅｉｍｍｕｎｉｔｙｃｏｎｔｉｎｕ…     

Expression and immunogenicity of recombinant Mycobacterium bovis Bacillus Calmette-Guérin strains secreting the antigen ESAT-6 from Mycobacterium tuberculosis in mice

Background Tuberculosis remains the leading cause of human death. Currently, Bacillus Calmette-Guérin (BCG) is the only available vaccine against tuberculosis but its efficacy is highly variable. Thus, developing new tuberculosis vaccines becomes an urgent task. In this study, we evaluated in BALB/c mice the humoral and cellular immune responses of recombinant BCG expressing the antigen ESAT-6 from Mycobacterium tuberculosis.Methods Escherichia coli-BCG shuttle plasmid named pDE22-esat-6 was constructed by inserting the BamHI/EcoRI digested esat-6 gene PCR product into the similarly digested parental plasmid pDE22. BCG cells were transformed with pDE22-esat-6, which was named recombinant BCG (rBCG). BALB/c mice were immunized subcutaneously on the back with 100 µl normal saline containing 10(6) CFU of BCG or rBCG. They were sacrificed after 4 weeks to detect their humoral and cellular responses. Results There was no any significant differences in the growth characteristics between the conventional BCG and rBCG. In immunized mice, the IgG antibody titres of rBCG group were as high as 1:8000, which was significantly higher than that in BCG group (1:1400, P<0.05). The elicited IFN-γ level of rBCG group was (1993 ± 106) pg/ml, which was also significantly higher than that in BCG group ((1463 ± 105) pg/ml, P<0.05). The splenocyte proliferation index of rBCG group reached 4.34 ± 0.31, which was higher than that of BCG group (3.79 ± 0.24, P<0.05).Conclusion rBCG secreted expressing antigen ESAT-6 stimulated stronger humoral and cellular immune responses than BCG did, and, therefore may be the better vaccine against mycobacterium tuberculosis.     

Construction and Expression of DNA Vaccine pIRES-Sj97-Sj14-Sj26 and Its Immunogenicity in Mice

To find a new preventive strategy for the infection of Schistosoma japonica, plasmid pIRES-Sj97-Sj 14-Sj26 that contains fatty binding protein （Sj 14）, GST （Sj26） and paramyocin （Sj97） that are expressed on the membrane, was constructed. RT-PCR was used to detect the expression of Sj 14 mRNA, Sj26 mRNA and Sj97 mRNA in the Hela cells, the indirect immunofluorescent test was employed for the detection of the expression of trans-membrane Sj26 after the plasmid was transfected into Hela cells. Fifty BALB/c mice were randomly divided into 5 groups and plRES-Sj97-SjI4-Sj26 plasmid DNA, plRES-Sj 14-Sj26 plasmid DNA, plRES-Sj26 plasmid DNA, plRES blank vector and normal saline were respectively injected into the quadriceps muscles of thigh Eight weeks after the immunization the mice were killed and significantly higher level of IgG was detected in the plRES-Sj97-Sj 14-Sj26 group as compared with the plRES blank vector, normal saline and plRES-Sj26 groups （P〈 0.01） and the plRES-Sj 14-Sj26（P〈0.05）. Single splenocyte suspension was prepared to detected the level of IFN-T by ELISA and the lymphocyte stimulating index （SI） by MTT. SI was significantly higher of in the plRES-Sj97-Sj 14-Sj26 group than in other groups （P〈 0.01）, while the IFN-T level was significantly higher the plRES-Sj97-Sj 14-Sj26 group than in plRES blank vector and normal saline groups （P〈0.01）, but no significant differences were found when compared with plRES-Sj 14-Sj26 and plRES-Sj26 groups. Flow cytometery showed that the percentages of CD4＋ and CD8＋ T cells were much higher in the plRES-Sj97-Sj 14-Sj26 group （P〈 0.01, P〈0.05）. It was concluded that plRES-Sj97-Sj 14-Sj26 vaccine may induce stronger immune response in BALB/c mice.     

FQ2在日本血吸虫DNA疫苗诱导免疫机制的研究

目的 探讨佐剂FQ2与日本血吸虫DNA26Kda疫苗共同作用下小鼠的免疫状态。方法 选取4～6周龄BALB/C小鼠52只，分成生理盐水组、FQ2组（A）、Sj26GST组（B）和FQ2＋Sj26GST组（C）等4组，于第0、2、6周各免疫1次，接种后4周以日本血吸虫尾蚴攻击感染，感染后6W剖杀小鼠，分析脾脏T淋巴细胞CD4^＋和CD8^＋细胞率，测定血清中特异性IgG水平。结果 B组和C组均可诱导CD8^＋淋巴细胞的增殖。C组升高更为显著，CD4^＋/CD8^＋比率倒置，也均可于4周后诱导小鼠产生高滴度的IgG抗体，二组间差异无显著（P〉0．05）。结论 FQ2对Sj26GST DNA疫苗有明显的增效作用，推测增效作用主要由细胞免疫所介导。     

Construction and expression of DNA vaccine pIRES-Sj97-Sj14-Sj26 and its immunogenicity in mice

Summary To find a new preventive strategy for the infection of Schistosoma japonica, plasmid pIRES-Sj97-Sj14-Sj26 that contains fatty binding protein (Sj14), GST (Sj26) and paramyocin (Sj97) that are expressed on the membrane, was constructed. RT-PCR was used to detect the expression of Sj14 mRNA, Sj26 mRNA and Sj97 mRNA in the Hela cells, the indirect immunofluorescent test was employed for the detection of the expression of trans-membrane Sj26 after the plasmid was transfected into Hela cells. Fifty BALB/c mice were randomly divided into 5 groups and pIRES-Sj97-Sj14-Sj26 plasmid DNA, pIRES-Sj14-Sj26 plasmid DNA, pIRES-Sj26 plasmid DNA, pIRES blank vector and normal saline were respectively injected into the quadriceps muscles of thigh. Eight weeks after the immunization the mice were killed and significantly higher level of IgG was detected in the pIRES-Sj97-Sj14-Sj26 group as compared with the pIRES blank vector, normal saline and pIRES-Sj26 groups (P<0.01) and the pIRES-Sj14-Sj26(P<0.05). Single splenocyte suspension was prepared to detected the level of IFN-γ by ELISA and the lymphocyte stimulating index (SI) by MTT. SI was significantly higher of in the pIRES-Sj97-Sj14-Sj26 group than in other groups (P<0.01), while the IFN-γ level was significantly higher the pIRES-Sj97-Sj14-Sj26 group than in pIRES blank vector and normal saline groups (P<0.01), but no significant differences were found when compared with pIRES-Sj14-Sj26 and pIRES-Sj26 groups. Flow cytometery showed that the percent-ages of CD4+ and CD8+ T cells were much higher in the pIRES-Sj97-Sj14-Sj26 group (P< 0.01, P<0.05). It was concluded that pIRES-Sj97-Sj14-Sj26 vaccine may induce stronger immune response in BALB/c mice. This project was supported by grants from National Natural Sciences Foundation of China (No. 30471603).     

针对Toll样受体4基因的小发夹结构RNA对RAW264.7细胞炎症因子分泌的抑制作用

目的构建针对Toll样受体(TLR)4 mRNA的小发夹结构RNA(shRNA)真核表达载体pEGFP-siRNA/TLR4,检测该shRNA诱导的RNA干扰(RNAi)对脂多糖刺激RAW264.7细胞分泌炎症因子的抑制作用,以探讨针对TLR4基因的RNAi对RAW264.7细胞炎症反应的抑制作用.方法构建携带增强型绿色荧光蛋白(EGFP)及shRNA克隆位点的质粒pEGFP-H1/siRNA,运用网络工具siRNA Wizard(http//www.simawizard.com/)设计针对TLR4 mRNA的寡核苷酸片段.将其克隆入pEGFP-H1/siRNA,构建表达EGFP及TLR4-shRNA的真核表达质粒pEGFP-H1/TLR4-siRNA.运用脂质体转染技术将pEGFP-H1/TLR4-siRNA转染至培养的小鼠巨噬细胞系RAW264.7,观察细胞系中荧光蛋白的表达强度;再运用脂多糖刺激转染的RAW264.7细胞,运用ELISA方法检测该细胞系分泌炎症因子水平的变化.结果质粒pEGFP-H1/siRNA及pEGFP-H1/TLR4-siRNA分别用Bbs Ⅰ和MluⅠ酶切电泳后,前者出现4.9 kb和340 bp的的条带,后者出现5.0 kb和220 bp的条带,与实验设计的质粒长度一致,且测序证实克隆序列正确.将pEGFP-H1/TLR4-siRNA转染至RAW264.7细胞后,EGFP的表达率为50%±8%.用脂多糖刺激后,TLR4-SiRNA转染组细胞培养液TNF-α水平(2 h825 pg/ml±136 pg/ml;8 h2190 pg/ml±359 pg/ml)明显低于对照siRNA转染组(2 h1179 pg/ml±240 pg/ml;8 h4720 pg/ml±227 pg/ml,均P＜0.01).结论针对TLR4 mRNA的shRNA可能通过RNAi机制对脂多糖诱导的RAW264.7细胞炎症因子的分泌有明显的抑制作用.     

日本血吸虫重组BCG-Sj26GST疫苗免疫小鼠血清IgG动态观察及免疫保护力的研究

为检测血吸虫重组BCG-Sj26GST疫苗免疫小鼠血清IgG动态变化和免疫保护力,采用106CFU疫苗皮下1次和3次接种小鼠,接种后8周用日本血吸虫尾蚴攻击感染。感染后6周剖杀小鼠,计算减虫率和减卵率,同时设有BCG对照组。结果发现实验组免疫后血清IgG抗体迅速升高并持续于高水平;减虫率分别为39.74%和39.74%,减卵率分别为62.86%和55.62%,与对照组相比均有非常显著的差异（P<0.01）。而免疫3次和免疫1次小鼠血清IgG抗体水平及减虫率、减卵率均无显著差异。血吸虫重组BCG-Sj26GST疫苗皮下注射1次即能诱导小鼠较高水平的IgG抗体和免疫保护力,是一种比较理想的新型疫苗,值得进一步研究。