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刘潇, 李文桂, 罗广旭. 铜绿假单胞菌重组Bb-pGEX-OprI疫苗的构建及其保护力的研究[J]. 四川大学学报(医学版), 2018, 49(1): 13-17.
引用本文: 刘潇, 李文桂, 罗广旭. 铜绿假单胞菌重组Bb-pGEX-OprI疫苗的构建及其保护力的研究[J]. 四川大学学报(医学版), 2018, 49(1): 13-17.
LIU Xiao, LI Wen-gui, LUO Guang-xu. Study on Construction of Recombinant Bb-pGEX-OprI Vaccine of Pseudomonas aeruginosa and Its Protection Effect[J]. Journal of Sichuan University (Medical Sciences), 2018, 49(1): 13-17.
Citation: LIU Xiao, LI Wen-gui, LUO Guang-xu. Study on Construction of Recombinant Bb-pGEX-OprI Vaccine of Pseudomonas aeruginosa and Its Protection Effect[J]. Journal of Sichuan University (Medical Sciences), 2018, 49(1): 13-17.

铜绿假单胞菌重组Bb-pGEX-OprI疫苗的构建及其保护力的研究

Study on Construction of Recombinant Bb-pGEX-OprI Vaccine of Pseudomonas aeruginosa and Its Protection Effect

  • 摘要: 目的 构建并鉴定含有铜绿假单胞菌(Pseudomonas aeruginosa, Pa)外膜蛋白I(OprI)基因与两歧双歧杆菌(Bifidobacterium bifidum, Bb)疫苗 (Bb-pGEX-OprI),并研究该疫苗对小鼠Pa感染的保护作用。方法 PCR扩增OprI抗原编码基因并将其定向克隆至pGEX-1λT,构建重组质粒pGEX-OprI,将pGEX-OprI电穿孔转化Bb,构建Bb-pGEX-OprI疫苗,进行双酶切、PCR和测序鉴定后,异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,SDS-PAGE和Western blot分别分析并鉴定表达产物。21只小鼠随机分为3组,分别灌胃接种Bb-pGEX-OprI 疫苗、空载体疫苗(Bb-pGEX-1λT)和Bb。首次免疫后4周用PA01株攻击。攻击后2周处死小鼠取肺组织,计数肺组织的细菌菌落数。免疫前、首次免疫后4周和PA01株攻击后2周采小鼠静脉血,常规ELISA检测血清IgG及其亚类和IgE。结果 PCR成功扩增出194 bp的OprI抗原编码基因;双酶切、PCR和测序证实OprI基因成功克隆入pGEX-1λT中,并且pGEX-OprI成功转化Bb,构建为Bb-pGEX-OprI疫苗;SDS-PAGE显示Bb-pGEX-OprI 表达相对分子质量约32×103的OprI-谷胱甘肽转移酶(GST)融合蛋白;Western blot证实融合蛋白能被Pa感染的鼠血清特异性识别。Bb-pGEX-OprI疫苗组小鼠肺组织的细菌菌落数低于Bb-pGEX-1λT组和Bb组(P<0.01),Bb-pGEX-OprI疫苗组小鼠血清IgG、IgG2b、IgG3和IgE水平在首次免疫后4周和攻击后2周依次升高,相同时间点Bb-pGEX-OprI疫苗组小鼠血清抗体水平均高于Bb-pGEX-1λT和Bb组(P<0.01或P<0.05)。结论 成功构建了重组疫苗Bb-pGEX-OprI,其在小鼠抗Pa感染过程中可产生有效的体液免疫应答。

     

    Abstract: Objective To construct the recombinant Bb-pGEX-OprI vaccine of Pseudomonas aeruginosa (Pa) outer membrane protein I (OprI) and study its protection effect in mice against Pa. Methods The OprI gene was amplified by PCR, and cloned into pGEX-1λT to generate pGEX-OprI. The pGEX-OprI was transformed into Bifidobacterium bifidum(Bb) to construct recombinant Bb-pGEX-OprI vaccine by electroporation. After identification with double enzyme digestion, PCR and sequencing, the vaccine was then induced with IPTG, and its expression was analyzed and identified by SDS-PAGE and Western blot respectively. Twenty-one mice were randomly divided into 3 groups and vaccinated by intragastric administration with Bb-pGEX-OprI, Bb-pGEX-1λT and Bb respectively. All mice were challenged with PA01 strain at 4 weeks after the first vaccination. At 2 weeks after the challenge, mice were sacrificed to separate their lungs, and the numbers of bacterial colonies in lungs were counted. Venous blood was collected before vaccination, at 4 weeks after the first vaccination and 2 weeks after the challenge of PA01 strain. The serum IgG, IgG subclasses and IgE were detected by routine ELISA. Results The OprI gene of 194 bp was successfully amplified by PCR. Double enzyme digestion, PCR and sequencing confirmed that the OprI gene was successfully cloned into pGEX-1λT and pGEX-OprI was successfully transformed into Bb, constructing the Bb-pGEX-OprI vaccine. SDS-PAGE indicated that Bb-pGEX-OprI vaccine expressed an OprI-GST fusion protein with the relative molecular mass of approximately 32×103. Western blot verified that the fusion protein could be specifically identified by the sera of mice infected with Pa. The number of bacterial colonies in lung of Bb-pGEX-OprI vaccine group was lower than that of Bb-pGEX-1λT or Bb control (P<0.01). The levels of serum IgG, IgG2b, IgG3 and IgE in Bb-pGEX-OprI vaccine group rose at 4 weeks after the first vaccination and 2 weeks after the challenge successively. The levels of serum antibodies in Bb-pGEX-OprI vaccine group were higher than those in Bb-pGEX-1λT or Bb control at the same time point (P<0.01 or P<0.05). Conclusion The recombinant Bb-pGEX-OprI vaccine was successfully constructed and produced an effective humoral immune response against the Pa infection.

     

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