日本血吸虫重组两歧双歧杆菌(pGEX-Sj32)疫苗构建及鉴定

目的 构建和鉴定日本血吸虫重组两歧双歧杆菌(Bifidobacterium bifidum,Bb)pGEX-Sj32疫苗.方法 从重庆医科大学附属第一医院传染病寄生虫病研究所构建的重组大肠埃希菌BL21 (pET28α-Sj32)中抽提质粒pET28α-Sj32,通过PCR扩增Sj32抗原编码基因;将Sj32基因定向克隆至大肠埃希菌-双歧杆菌穿梭表达载体pGEX-1λT中,构建重组质粒pGEX-Sj32,将该重组质粒转化至大肠埃希菌BL21感受态细胞(DE3),抽提质粒后进行双酶切鉴定;采用电穿孔法,将重组质粒pGEX-Sj32转化至Bb,构建Bb(pGEX-Sj32)疫苗,再从该疫苗中抽提重组质粒pGEX-Sj32,以其为模板进行PCR鉴定.结果 从抽提质粒pET28α-Sj32中PCR扩增出长度约为1 270 bp的Sj32基因片段;重组质粒pGEX-Sj32经双酶切鉴定,获得长度为4 947 bp的载体片段和长度为1 270 bp的Sj32基因片段;以抽提的疫苗质粒pGEX-Sj32为模板进行PCR扩增,得到了长度约为1 270 bp的Sj32基因片段,与预期结果相符.结论 成功构建了日本血吸虫重组Bb(pGEX-Sj32)疫苗.     

日本血吸虫重组质粒pET28α-Sj26GST-Sj32的构建及其在大肠埃希菌BL21(DE3)中的表达

目的 构建日本血吸虫重组质粒pET28α-Sj26GST-Sj32,观察该质粒在大肠埃希菌BL21(DE3)中的表达.方法 超声粉碎日本血吸虫成虫,提取总RNA,通过RT-PCR扩增获得Sj26GST和Sj32抗原编码基因,然后采用基因拼接法剪接Sj26GST和Sj32,得到Sj26GST-Sj32融合基因,克隆至大肠埃希菌原核表达载体pET28α,构建重组质粒pET28α-Sj26GST-Sj32,转化大肠埃希菌BL21(DE3),经异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达后用十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)和Western印迹法对表达产物进行分析和鉴定.结果 基因拼接法扩增出长度约1991 bp的Sj26GST-Sj32融合基因;双酶切和PCR鉴定证实Sj26GST-Sj32融合基因成功插入pET28α中,SDS-PAGE分析显示表达产物为相对分子质量约69×103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25%;Western印迹法鉴定重组蛋白能被日本血吸虫感染的兔血清识别.结论 成功构建了日本血吸虫重组质粒pET28α-Sj26GST-Sj32,该质粒在大肠埃希菌BL21(DE3)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.

Abstract：

Objective To construct and express the recombinant plasmid pET28α-Sj26GST-Sj32 of Schistosoma japonicum(Sj) in Escherichia coli BL21 (DE3). Methods Total RNA was extracted from Sj adult worms by ultrasound-breaking, Sj26GST and Sj32 antigen gene was respectively amplified by RT-PCR from the total RNA; Sj26GST-Sj32 fusion gene obtained with gene splicing by overlap extension(SOEing) was cloned into prokaryotic expression plasmid pET28α and transformed into Escherichia coli BL2 (DE3) to construct pET28α-Sj26GST-Sj32;BL21 (pET28α-Sj26GST-Sj32) was induced with isopropyl-β-D-thiogalactopyranosid (IPTG), and the expressed products were analyzed and identified by sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE)and Western blotting. Results The 1991 bp Sj26GST-Sj32 fusion gene was successfully amplified by gene SOEing and cloned into pET28α by restriction analysis and PCR identification, the recombinant plasmid pET28α-Sj26GST-Sj32 was successfully constructed; the relative molecular mass of the expressed recombinant protein was approximately 69 × 103 by SDS-PAGE, and the amount of the expressed protein was 25% of the total bacterial proteins; the fusion protein could be recognized by sera from rabbits infected with Sj by Western blotting.Conclusions The recombinant plasmid pET28α-Sj26GST-Sj32 is successfully constructed and highly expressed in Escherichia coli in fused form with His-tag, and the expressed fusion protein shows specific antigenicity.     

日本血吸虫重组质粒pQE30-Sj26GST-Sj32的构建及其在大肠埃希菌BL21(DE3)中的表达

目的构建日本血吸虫重组质粒pQE30-Sj26GST-Sj32,研究该质粒在大肠埃希菌BL21(DE3)中的表达。方法超声粉碎日本血吸虫成虫,提取总RNA,通过RT-PCR扩增获Sj26GST和Sj32抗原编码基因;采用基因拼接法(gene SOEing)剪接Sj26GST和Sj32,得到Sj26GST-Sj32融合基因;将融合基因克隆至原核表达载体pQE30,构建重组质粒pQE30-Sj26GST-Sj32并转化大肠埃希菌BL21,经异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达后用SDS-PAGE和Western blot对表达产物进行分析和鉴定。结果基因拼接法扩增出约1 991 bp的Sj26GST-Sj32融合基因;双酶切和PCR鉴定证实Sj26GST-Sj32融合基因成功插入pQE30中,SDS-PAGE显示表达产物为分子质量单位约为61 ku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18%;Western blot鉴定重组蛋白能被日本血吸虫感染的兔血清识别。结论成功构建了日本血吸虫重组质粒pQE30-Sj26GST-Sj32,该质粒在大肠埃希菌BL21中获得了高效融合表达,表达的融合蛋白具有抗原特异性。     

日本血吸虫谷胱甘肽-S-转移酶重组质粒的构建及其在大肠埃希菌BL21(DE3)中的表达

目的 构建日本血吸虫谷胱甘肽-S-转移酶(Sj26GST)重组质粒pET32α-Sj26GST,观察该质粒在大肠埃希菌BL21(DE3)中的表达.方法 超声粉碎日本血吸虫成虫,提取总RNA,通过RT-PCR扩增Sj26GST抗原编码基因,克隆至原核表达载体pET32α(+),构建pET32α-Sj26GST,转化大肠埃希菌BL21(DE3),经异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达后,用十二烷基磺酸钠-聚丙烯酰氨凝胶电泳(SDS-PAGE)和Western blot 对表达产物进行分析和鉴定.结果 RT-PCR扩增出676 bp的Sj26GST基因;双酶切和PCR鉴定证实Sj26GST基因成功插入pET32α(+)中,SDS-PAGE分析显示表达产物为相对分子质量约为49×103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24%;Western blot鉴定重组蛋白能被日本血吸虫感染的兔血清识别.结论 成功构建了pET32α-Sj26GST,该质粒在大肠埃希菌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.     

日本血吸虫重组两歧双歧杆菌pGEX-Sj14-3-3疫苗构建及鉴定

目的 构建日本血吸虫重组双歧杆菌属两歧双歧杆菌(Bifidobacterium bifidum,Bb)pGEx-Sj14-3-3疫苗,并进行鉴定.方法 从日本血吸虫成虫中提取总RNA,通过RT-PCR扩增Sj14-3-3抗原编码基因.将Sj14-3-3基因定向克隆到大肠埃希菌-双歧杆菌穿梭表达载体pGEX-1λT中,构建重组质粒pGEX-Sj14-3-3.用重组质粒将大肠埃希菌BL21(DE3)转化为感受态细胞,抽提重组质粒进行双酶切,鉴定载体片段和基因片段长度:采用电穿孔法,用重组质粒pGEX-Sj14-3-3转化Bh,构建Bb(pGEX-Sj14-3-3)疫苗,抽提疫苗的质粒进行PCR鉴定,比较重新构建的pGEX-Sj14-3-3基因片段长度与日本血吸虫成虫中Sjl4-3-3基因片段长度是否相同.结果 RT-PCR扩增出的日本血吸虫成虫Sj14-3-3基因片段长度为399 bp;双酶切鉴定,重组质粒pGEX-Sj14-3-3载体片段长度为4947 bp,Sj14-3-3基因片段长度为399 bp;在重组的Bb(pGEX-Sj14-3-3)疫苗中,得到Sj14-3-3基因片段长度为399 bp,与预期的结果一致.结论 成功构建了日本血吸虫重组Bb(pGEX-Sj14-3-3)疫苗.

Abstract：

Objective To construct and identify recombinant vaccine Bifwlobacterium bifidum(Bb)pGEX-Sj14-3-3 of Schistosoma japonicum(Sj). Methods Total RNA was extracted from adult Sj, antigen encoding gene Sj14-3-3 was amplified by RT-PCR and cloned into Escherichia coli (E. coli)-Bb shuttle expression vector pGEX-1λT to construct recombinant plasmid pGEX-Sj14-3-3. The recombinant plasmid was transformed into E. coli BL21 (DE3).The plasmid was extracted and identified by using BamH I and EcoR I. Then pGEX-Sjl4-3-3 was electroporated into Bb to construct recombinant Bb (pGEX-Sj14-3-3) vaccine. The extracted plasmid of the recombinant Bb (pGEX-Sj14-3-3) vaccine was identified by PCR, and the size of the products was compared with Sj14-3-3 gene of adult worms.Results Sj14-3-3 of 399 bp in length was amplified by RT-PCR. The products were digested by BamH I and EcoR I , and the fragments length of plasmid pGEX-Sj14-3-3 vector was 4947 bp, and of Sj 14-3-3 gene was 399 bp.The product of 399 bp Sj14-3-3 gene was also amplified by PCR from template of the extracted plasmid of the recombinant Bb(pGEX-Sj14-3-3 ) vaccine. The size of the product obtained was just the same as expected.Conclusion The recombinant Bb(pGEX-Sj14-3-3) vaccine of Sj is successfully constructed.     

日本血吸虫重组质粒pET28α-Sj32的构建及其在大肠埃希菌BL21(DE3)中的表达

目的构建日本血吸虫重组质粒pET28α-Sj32,研究该质粒在大肠埃希菌BL21(DE3)中的表达。方法超声粉碎日本血吸虫成虫,提取总RNA,通过RT-PCR扩增Sj32抗原编码基因;将该基因克隆至原核表达载体pET28α,构建重组质粒pET28α-Sj32并转化大肠埃希菌BL21(DE3),经异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达后用SDS-PAGE和Western blot对表达产物进行分析和鉴定。结果RT-PCR扩增出1270bp的Sj32基因;双酶切和PCR鉴定证实Sj32基因成功插入pET28α中,SDS-PAGE分析显示表达产物为分子质量单位约为42ku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24%;Western blot鉴定重组蛋白能被日本血吸虫感染的兔血清识别。结论成功构建了日本血吸虫重组质粒pET28α-Sj32,该质粒在大肠埃希菌BL21中获得了高效融合表达,表达的融合蛋白具有抗原特异性。     

幽门螺杆菌重组Bb-hpaA-vacA疫苗的构建

目的 构建幽门螺杆菌( Hp) 重组双歧杆菌( Bb) Bb-hpaA-vacA疫苗。方法 通过 PCR 分别扩增hpaA和vacA抗原编码基因, 然后采用gene SOEing 剪接hpaA和vacA, 得到hpaA-vacA融合基因;将该融合基因定向克隆到大肠埃希菌-双歧杆菌穿梭表达载体pGEX-1λT, 构建重组质粒 pGEX-hpaA-vacA,电穿孔法将该质粒导入Bb, 构建幽门螺杆菌重组hpaA-vacA 疫苗。结果 PCR成功扩增出分子量约为1500 bp的 hpaA-vacA融合基因,双酶切证实hpaA-vacA融合基因成功插入pGEX-1λT中,并成功转化入双歧杆菌,构建rBb-hpaA-vacA疫苗。结论 成功构建螺杆菌rBb-hpaA-vacA疫苗,为该疫苗的进一步研究奠定了基础。     

细粒棘球绦虫重组Bb-Eg95-EgA31融合基因疫苗构建及鉴定

目的构建和鉴定细粒棘球绦虫(Eg)重组双歧杆菌(Bb)-Eg95-EgA31融合基因疫苗。方法自行设计引物,从细粒棘球蚴包囊中分离原头节,超声粉碎后提取总RNA为模板,通过RT-PCR分别扩增Eg95和EgA31抗原编码基因,然后采用基因拼接法(gene SOEing)剪接Eg95和EgA31,得到Eg95-EgA31融合基因,经BamHⅠ和EcoRⅠ双酶切,定向克隆到大肠杆菌-双歧杆菌穿梭表达载体pGEX-1λT中,转化大肠杆菌BL21(DE3)感受态细胞,构建重组质粒pGEX-Eg95-EgA31,抽提质粒进行双酶切鉴定,电穿孔法转化两歧双歧杆菌(Bifidobacteria bifidum,Bb),构建细粒棘球绦虫重组Bb-Eg95-EgA31融合基因疫苗,抽提质粒进行PCR扩增鉴定。结果RT-PCR扩增出约1 016bp的Eg95-EgA31融合基因;重组质粒用双酶切鉴定可切出预期大小片段,以具有氨苄青霉素抗性的rBb中抽提的质粒为模板进行PCR扩增可得到约1016bp的Eg95-EgA31融合基因片段。结论成功构建了细粒棘球绦虫重组Bb-Eg95-EgA31融合基因疫苗,为该疫苗的开发利用奠定了实验基础。     

细粒棘球绦虫重组Bb-Eg95疫苗的构建及鉴定

目的 构建和鉴定细粒棘球绦虫(Eg)重组双歧杆菌(rBb)-Eg95疫苗.方法 自行设计引物,从细粒棘球蚴包囊中分离原头节,超声粉碎后提取总RNA为模板,通过RT-PCR扩增获得Eg95抗原编码基因,采用BamH Ⅰ和EcoR Ⅰ双酶切,定向克隆到大肠埃希菌-双歧杆菌(E.coli-Bb)穿梭表达载体pGEX-1λT中,转化E.coli BL21(DE3)感受态细胞,构建重组质粒pGEX-Eg95.抽提质粒进行双酶切鉴定后,电穿孔转化到Bb中,构建细粒棘球绦虫rBb-Eg95疫苗,抽提质粒进行PCR扩增鉴定.结果 RT-PCR扩增出471 bp的Eg95抗原编码基因;重组质粒用双酶切鉴定可切出4947 bp的载体片段和471 bp的目的 基因片段:以具有氨苄青霉素抗性的rBb中抽提的质粒为模板进行PCR扩增可得到471 bp的Eg95基因片段.结论 成功构建了细粒棘球绦虫rBb-Eg95疫苗,为该疫苗的开发利用奠定了理论基础.     

铜绿假单胞菌重组Bb-OprF疫苗构建及鉴定

目的构建和鉴定铜绿假单胞菌重组双歧杆菌(rBb) OprF疫苗。方法以铜绿假单胞菌PAOl标准株提取总RNA为模板,自行设计引物,RT PCR扩增获得OprF抗原编码基因,经酶切、连接定向克隆入大肠埃希菌 双歧杆菌穿梭表达载体pGEX 1λT,构建重组质粒pGEX OprF。转化E.coli BL21(DE3)感受态细胞,抽提质粒行酶切电泳和测序验证后,电穿孔转化到Bb中,构建铜绿假单胞菌rBb OprF疫苗,抽提质粒进行PCR扩增鉴定。结果RT PCR扩增出1 016bp的OprF编码基因;重组质粒经双酶切鉴定,切出4 947bp的载体片段和10 16bp的目的基因片段;以rBb抽提质粒为模板进行PCR扩增可得到1 016bp的oprF基因片段。结论成功构建了铜绿假单胞菌rBb OprF疫苗,为该疫苗的进一步研究奠定基础。