表达PCV2 Cap蛋白减毒鼠伤寒沙门氏菌活载体疫苗的构建及动物免疫试验
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摘要
本研究目的旨在构建一种表达PCV2 Cap蛋白减毒鼠伤寒沙门氏菌活载体疫苗,通过动物免疫试验对构建的疫苗进行评价。Cap蛋白是PCV2 ORF2基因编码的主要结构蛋白和免疫保护性蛋白,本研究选择截去N端41个氨基酸的核定位区的ORF2编码基因作为目的基因,利用减毒鼠伤寒沙门氏菌的平衡致死系统表达优化的ORF2基因,构建表达Cap蛋白的活载体疫苗,并对重组菌苗的生物学特性和免疫学特性进行评价。研究取得如下结果:
1表达猪圆环病毒Cap蛋白的鼠伤寒沙门氏菌活载体疫苗的构建
合成特异性引物P1、P2,PCR扩增得到ORF2基因,将其插入经BamH I和Pst I双酶切的Asd+的组成型表达载体pYA3341中,构建重组表达载体pYA3341-ORF2;重组载体电转化入缺失天冬氨酸-β-半醛脱氢酶(Δasd)、腺苷酸环化酶(Δcya)以及环腺苷酸受体蛋白基因(Δcrp)的减毒鼠伤寒沙门氏菌中间宿主菌X3730及终末宿主菌X4550中,获得重组活载体疫苗菌X4550(pYA3341-ORF2)。
2重组活载体疫苗菌的生物学特性检测
研究重组活载体疫菌苗X4550(pYA3341-ORF2)体外稳定特性、生长特性、口服安全性及在小鼠体内的定值分布情况。结果显示,Cap蛋白能在X4550中有高水平的表达,且重组菌能在体外稳定遗传、生长状态良好、口服安全无毒性及在机体中具有良好的定值分布特性。
3重组活载体疫苗菌的免疫原性研究
将重组活载体疫苗菌X4550(pYA3341-ORF2)口服免疫Balb/C小鼠作为免疫组,以口服重组菌X4550(pYA3341)的小鼠作为阴性对照组,以口服PBS的小鼠作为空白对照组。ELISA方法检测疫苗免疫组小鼠血清抗体效价达到1:1920;间接免疫荧光法检测疫苗免疫组小鼠血清中和抗体效价可达到1:19.2;MTT法检测免疫组小鼠淋巴细胞分裂指数可达到4.26;流式细胞术检测疫苗免疫组小鼠的CD4+、CD8+T淋巴细胞数目分别约为29.9、7.52,CD4+/CD8+T细胞比值约为3.99。将重组活载体疫苗菌X4550(pYA3341-ORF2)免疫猪,ELISA法检测疫苗免疫猪血清抗体效价1:57.6;疫苗免疫组猪血清的中和抗体效价可达到1:11.2;疫苗免疫组淋巴细胞分裂指数可达到3.82。
重组菌苗X4550(pYA3341-ORF2)能够稳定表达Cap蛋白,具有良好免疫原性,为该疫苗的进一步研制与临床应用提供参考依据。
This study aimed to construct a kind of live vector vaccine which could express Cap protein of PCV2 by attenuated Salmonella typhimurium(S.t), and evaluated the live vector vaccine by animals immunization. Capsid protein was encoded by ORF2 gene of PCV2, and this protein has been identified as the major structural and immunogenic antigen of PCV2. Therefore, the ORF2 gene deleted the 41 amino acid was chosen as an aimed gene in this study. Utilizing the balanced lethal symtem of attenuated Salmonella typhimurium to express the optimization of the ORF2 gene, construct live vector vaccine expressed Cap protein, and to evaluate biological traits and immunological properties of the vaccine strain. Some results were as follows:
1 Construction of attenuated S.t live vector vaccine expressing Cap protein of PCV2
Specificity primers P1 and P2 was synthesized and the optimization of the ORF2 gene was amplified, then the ORF2 gene was inserted into the attenuated S.t expression vector pYA3341 containing asd gene by double digestion of BamH I and Pst I, and constructed the recombinant expression plasmid pYA3341-ORF2.The reconstructed plasmid was electrotransformed into a balanced-lethal recombinant attenuated S.t strain X3730 and X4550 with one by one, which were deltd aspartate-β-semialdehyde dehydrogenase (ΔAsd), adenylyl cyclase (ΔCya) and cyclic AMP receptor protein gene (ΔCrp). In the end, we got oral vaccine strain X4550 (pYA3341-ORF2).
2 Detection of the biological characteristics of recombinant vaccine strain
The vitro stablity, growth characteristic, orally safety of recombinant vaccine strain X4550(pYA3341-ORF2), and distribution in mice after orally administered of the recombinant vaccine were also analyzed. The results showed that the Cap protein in the X4550 had a high level of expression, and the recombinant strain had good genetic stability in vitro, growth states, safty, non-toxic and well distribution in the body.
3 Research of the immunogenicity of recombinant vaccine strain
Treatment group was immunized intragastrically with recombinant vaccine strain X4550 (pYA3341-ORF2), negative control group were immunized intragastrically with recombinant strain X4550 (pYA3341) and control group were taken orally with PBS. Antibody titer of serum of treated mice can reach to 1:1920 by ELISA; Neutralization activity of the treated mice serum can reach to 1:19.2 by indirect immunofluorescence; Stimulation index of lymphocyte of the treated group was 4.26 by MTT; The CD4+ and CD8+ lymphocyte subsets populations of immuned mice were separately 29.9, 7.52 by flow cytometry, and the ratio of CD4+/CD8+ was 3.99. Then the piglets were also immunized intragastrically with recombinant vaccine strain X4550 (pYA3341-ORF2), negative control group were immunized intragastrically with recombinant strain X4550 (pYA3341) and control group were taken orally with PBS. Antibody titer of serum of treated piglets was 1:57.6 by ELISA; Neutralization activity of the serum was 1:11.2 by IFA; Stimulation index of lymphocyte of the treated group was 3.82 by MTT.
Above studies suggested that the recombinant vaccine strain can express stably Cap protein of PCV2, which had good immunogenicity. This would supply some dates for further develop of vaccine and clinical application in the nearly future.
1表达猪圆环病毒Cap蛋白的鼠伤寒沙门氏菌活载体疫苗的构建
合成特异性引物P1、P2,PCR扩增得到ORF2基因,将其插入经BamH I和Pst I双酶切的Asd+的组成型表达载体pYA3341中,构建重组表达载体pYA3341-ORF2;重组载体电转化入缺失天冬氨酸-β-半醛脱氢酶(Δasd)、腺苷酸环化酶(Δcya)以及环腺苷酸受体蛋白基因(Δcrp)的减毒鼠伤寒沙门氏菌中间宿主菌X3730及终末宿主菌X4550中,获得重组活载体疫苗菌X4550(pYA3341-ORF2)。
2重组活载体疫苗菌的生物学特性检测
研究重组活载体疫菌苗X4550(pYA3341-ORF2)体外稳定特性、生长特性、口服安全性及在小鼠体内的定值分布情况。结果显示,Cap蛋白能在X4550中有高水平的表达,且重组菌能在体外稳定遗传、生长状态良好、口服安全无毒性及在机体中具有良好的定值分布特性。
3重组活载体疫苗菌的免疫原性研究
将重组活载体疫苗菌X4550(pYA3341-ORF2)口服免疫Balb/C小鼠作为免疫组,以口服重组菌X4550(pYA3341)的小鼠作为阴性对照组,以口服PBS的小鼠作为空白对照组。ELISA方法检测疫苗免疫组小鼠血清抗体效价达到1:1920;间接免疫荧光法检测疫苗免疫组小鼠血清中和抗体效价可达到1:19.2;MTT法检测免疫组小鼠淋巴细胞分裂指数可达到4.26;流式细胞术检测疫苗免疫组小鼠的CD4+、CD8+T淋巴细胞数目分别约为29.9、7.52,CD4+/CD8+T细胞比值约为3.99。将重组活载体疫苗菌X4550(pYA3341-ORF2)免疫猪,ELISA法检测疫苗免疫猪血清抗体效价1:57.6;疫苗免疫组猪血清的中和抗体效价可达到1:11.2;疫苗免疫组淋巴细胞分裂指数可达到3.82。
重组菌苗X4550(pYA3341-ORF2)能够稳定表达Cap蛋白,具有良好免疫原性,为该疫苗的进一步研制与临床应用提供参考依据。
This study aimed to construct a kind of live vector vaccine which could express Cap protein of PCV2 by attenuated Salmonella typhimurium(S.t), and evaluated the live vector vaccine by animals immunization. Capsid protein was encoded by ORF2 gene of PCV2, and this protein has been identified as the major structural and immunogenic antigen of PCV2. Therefore, the ORF2 gene deleted the 41 amino acid was chosen as an aimed gene in this study. Utilizing the balanced lethal symtem of attenuated Salmonella typhimurium to express the optimization of the ORF2 gene, construct live vector vaccine expressed Cap protein, and to evaluate biological traits and immunological properties of the vaccine strain. Some results were as follows:
1 Construction of attenuated S.t live vector vaccine expressing Cap protein of PCV2
Specificity primers P1 and P2 was synthesized and the optimization of the ORF2 gene was amplified, then the ORF2 gene was inserted into the attenuated S.t expression vector pYA3341 containing asd gene by double digestion of BamH I and Pst I, and constructed the recombinant expression plasmid pYA3341-ORF2.The reconstructed plasmid was electrotransformed into a balanced-lethal recombinant attenuated S.t strain X3730 and X4550 with one by one, which were deltd aspartate-β-semialdehyde dehydrogenase (ΔAsd), adenylyl cyclase (ΔCya) and cyclic AMP receptor protein gene (ΔCrp). In the end, we got oral vaccine strain X4550 (pYA3341-ORF2).
2 Detection of the biological characteristics of recombinant vaccine strain
The vitro stablity, growth characteristic, orally safety of recombinant vaccine strain X4550(pYA3341-ORF2), and distribution in mice after orally administered of the recombinant vaccine were also analyzed. The results showed that the Cap protein in the X4550 had a high level of expression, and the recombinant strain had good genetic stability in vitro, growth states, safty, non-toxic and well distribution in the body.
3 Research of the immunogenicity of recombinant vaccine strain
Treatment group was immunized intragastrically with recombinant vaccine strain X4550 (pYA3341-ORF2), negative control group were immunized intragastrically with recombinant strain X4550 (pYA3341) and control group were taken orally with PBS. Antibody titer of serum of treated mice can reach to 1:1920 by ELISA; Neutralization activity of the treated mice serum can reach to 1:19.2 by indirect immunofluorescence; Stimulation index of lymphocyte of the treated group was 4.26 by MTT; The CD4+ and CD8+ lymphocyte subsets populations of immuned mice were separately 29.9, 7.52 by flow cytometry, and the ratio of CD4+/CD8+ was 3.99. Then the piglets were also immunized intragastrically with recombinant vaccine strain X4550 (pYA3341-ORF2), negative control group were immunized intragastrically with recombinant strain X4550 (pYA3341) and control group were taken orally with PBS. Antibody titer of serum of treated piglets was 1:57.6 by ELISA; Neutralization activity of the serum was 1:11.2 by IFA; Stimulation index of lymphocyte of the treated group was 3.82 by MTT.
Above studies suggested that the recombinant vaccine strain can express stably Cap protein of PCV2, which had good immunogenicity. This would supply some dates for further develop of vaccine and clinical application in the nearly future.
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