减毒沙门氏菌递呈PEDV/TGEV双基因核酸疫苗研究
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摘要
猪流行性腹泻病毒(PEDV)和猪传染性胃肠炎病毒(TGEV)主要引起猪的病毒性腹泻,这两种病原引起的腹泻对2周龄内新生仔猪危害尤为严重,可导致感染仔猪发生严重的腹泻、呕吐与脱水,死亡率高达100%,给养猪业造成严重的经济损失。如何增强猪的肠道特异性黏膜免疫对其防治具有重要意义。本研究在临床分离鉴定PEDV的基础上,基于PEDV和TGEV相似的致病机理和免疫特点,创新性研究增强黏膜免疫诱导效力的新型口服基因疫苗的免疫机理,以PEDV S基因及TGEV S双基因为靶基因,pVAXD双启动子真核表达质粒为载体,进行减毒沙门氏菌携带的PEDV和TGEV二联DNA疫苗的构建与免疫原性研究,主要研究内容如下：
1.猪流行性腹泻病毒的分离鉴定
本文用Vero细胞成功地从仔猪腹泻粪样中分离了PEDV四川分离株(SC-L株),并对其生物学特性进行了初步研究。PEDV SC-L在Vero细胞上连续传代,增殖稳定,典型病变在接毒后48小时开始出现,表现为细胞变圆、细胞肿胀、最终细胞皱缩、破碎并脱落。通过透射电镜观察发现：病毒粒子在感染细胞胞浆内可见散在或呈晶格状排列的空心或实心的病毒粒子；病毒粒子成堆或成串排列在细胞膜上,与细胞膜融合,然后以胞外分泌的方式被释放到细胞膜外。病毒对5-溴脱氧尿核苷不敏感,是一种RNA病毒,对氯仿、乙醚敏感,60℃或以上处理30min便会失去感染力。通过对其S基因S1区进行序列测定,发现与韩国DR13株同源性最高,核苷酸同源性高达99.2%。遗传进化树分析显示所分离毒株与韩国DR13分离株亲缘关系最近。该毒株的成功分离为该病的诊断和防制积累了基础材料。
2. PEDV S基因主要抗原区域基因片断的克隆及原核表达研究
采用PCR方法从PEDV S基因TA克隆质粒中扩增出S基因的主要抗原区域基因片段Sa,扩增得到的Sa片段插入到pMD19-T simple中,构建的重组质粒命名为pMD19-T-Sa。对核苷酸序列进行测定和分析：扩增的基因长度为739bp,可编码247个氨基酸残基。本研究将Sa插入pET-32a(+)原核表达质粒,构建了原核表达质粒pET-32a(+)-PEDV-Sa,在大肠杆菌BL21中进行原核表达,SDS-PAGE电泳鉴定,原核表达质粒pET-32a(+)-PEDV-Sa表达的目的融合蛋白大小为45Kd,表达的蛋白经亲和层析法纯化后免疫家兔,制备PEDV Sa重组蛋白的多克隆抗血清。
3. PEDV S基因与TGEV S基因双基因共表达真核质粒的构建
采用RT-PCR扩增PEDV S基因S1区(包含病毒的主要中和表位和受体结合区域)和TGEV SC-H株S基因(包含A、B、C、D四个抗原位点),分别将其插入pMD19-T simple载体,构建重组质粒pMD19-T-PSl与pMD19-T-TS。对pMD19-T-PS1与pMD19-T-TS的核苷酸序列进行测定：克隆的PEDV SC-L株S1基因长约2367bp,编码789个氨基酸残基,TGEV SC-H株S基因长约1896bp,编码632个氨基酸残基。序列比对分析显示：PEDV SC-L株S1基因编码的氨基酸序列与其它PEDV毒株的同源性在88.8%-98.6%,TGEV SC-H株与其它TGEV毒株S基因编码的氨基酸序列同源性在95.5%～99.7%之间。研究以pVAXD双启动子真核表达质粒为载体,构建了能够同时表达PEDV S1和TGEV S基因的真核表达质粒pVAXD-PS1-TS以及分别单独表达PEDV S1基因和TGEV S基因的真核表达质粒pVAXD-PS1和pVAXD-TS。将构建成功的三种真核表达质粒以脂质体法分别转染COS-7细胞,通过间接免疫荧光检测发现：真核表达质粒转染的COS-7细胞在倒置荧光显微镜下呈现特异性荧光；表明上述构建的三种重组质粒可在体外表达。PEDV S1基因与TGEV S基因真核表达质粒pV AXD-PS1-TS、pV AXD-PS1与pVAXD-TS的成功构建为PEDV和TGEV DNA疫苗的进一步研究奠定了基础。
4.携带PEDV S和TGEV S双基因的减毒沙门氏菌口服疫苗免疫研究
研究将三种真核表达质粒pV AXD-PS1-TS、pV AXD-PS1与pVAXD-TS分别电转化入减毒鼠伤寒沙门氏菌SL7207中,构建了同时携带PEDV S1和TGEV S基因的重组减毒沙门氏菌SL7207(pVAXD-PS1-TS);以及携带PEDV S1基因的重组减毒沙门氏菌SL7207(pVAXD-PS1)和携带TGEV S基因的重组减毒沙门氏菌SL7207(pVAXD-TS)。小鼠灌胃接种重组减毒沙门氏菌后,提取小鼠肠道细胞总RNA,通过RT-PCR检测S1与S基因的转录。将重组菌SL7207(pVAXD-PS1)与SL7207(pVAXD-TS)混合以1×109CFU/只的剂量灌胃接种小鼠,SL7207(pVAXD-PS1-TS)以1×109CFU/只的剂量灌胃接种,间接ELISA方法检测免疫后小鼠的特异性PEDV和TGEV血清IgG以及肠道IgA抗体水平。结果：重组减毒沙门氏菌可有效激发免疫小鼠特异性PEDV(?)TGEV血清IgG以及肠道IgA抗体的产生,具有良好的免疫原性。通过比较单基因混合免疫组和双基因免疫组之间的抗体水平进一步表明：单基因混合免疫组的特异性血清IgG与肠道IgA抗体在三个免疫组中最高,且在免疫后第六周显著高于(P<0.05)其它免疫组。研究为PEDV和TGEV口服DNA疫苗的研究和应用奠定了基础。
Porcine Epidemic Diarrhea Virus (PEDV) and Transmissible Gastroenteritis Virus (TGEV) are major factor of swine viral diarrhea, and diarrhea of newborn piglets less than2weeks old caused by these two pathogens is particularly serious, which leads to severe vomiting, diarrhea and dehydration on infected piglets and nearly100%mortality rate, resulting serious economic losses for the swine industry. Thus, the enhancement of swine's specific enteric mucosal immunity plays a vital role in the prevention and therapy for both PEDV and TGEV. Based on the clinically isolated and then identified PEDV, the shared pathogenic mechanism and immune characteristics of PEDV and TGEV are taken into consideration to study on the the pathogenic mechanism of the innovatively contrusted oral DNA vaccine that could enhance mucosal immunity efficiency:PEDV S gene and TGEV S gene are used as target genes and inserted into dual promoter of eukaryotic expression vector pVAXD to conduct and study the immunogenicity of PEDV and TGEV bivalent DNA vaccine delivered by attenuated Salmonella typhimurium SL7207. The main contents were as follows:
1. Isolation and identification of Porcine Epidemic Diarrhea Virus (PEDV)
In this study, PEDV Sichuan strain SC-L was successfully isolated from diarrhea fecal samples via Vero cells, which followed by preliminary study on its biological characteristics. The proliferation of PEDV SC-L on Vero cells was stable during serial passages and typical lesions appeares48hours post-inoculation, the cells get round, swell, and ultimately shrink, splid, and shed from the culture plate. Observed by transmission electron microscopy, the hollow or solid virus particles are scattered or in lattice-like arrangement in infected intracytoplasm; the virus particles pile or cluster on the cell membrane, fusing with the cell membrane and secreting outside the cell membrane. This RNA virus is insensitive to5-bromodeoxyuridine urinary nucleosides but sensitive to chloroform and aether, it loses infectbility when in the circumstance of60℃or above for 30min. By Sequencing S1domain of S gene, it is in the highest homology with South Korea DR13strains with nucleotide homology high up to99.2%. The phylogenetic tree analysis shows that the isolated strains and South Korea DR13isolated strains are in the closest genetic relationship. This successfully isolated strain accounts for material foundation of diagnosis and prevention for PEDV.
2. Cloning and prokaryotic expression of PEDV S gene main antigtic domain fragment
The PEDV S gene main antigtic domain fragment Sa were amplified from the PEDV S gene TA cloning plasmid via PCR and then cloned into the pMD19-T simple vector to construct the recombinant plasmid pMD19-T-Sa. The amplified gene is739bp in length and is able to encode247amino acids accroding to the sequence analysis. The recombinant prokaryotic plasmid pET-32a(+)-PEDV-Sa with Sa instered into pET-32a(+) prokaryotic expression plasmid could produce45Kd fusion protein in E.coli BL21, which is detected by SDS-PAGE electrophoresis. The expressed protein was purified by affinity chromatography immune and polyclonal antiserum was prepared by immunizing rabbits with this PEDV Sa recombinant protein.
3. The construction of PEDV S gene and TGEV S gene co-expression eukaryotic plasmid
The RT-PCR amplified S1region of PEDV S gene that contains main neutralization epitope and receptor binding region of the virus, and5'fragment of S gene of the TGVE SC-H strain that covers A and B, and C, and D antigens points were respectively cloned into pMD-19T simple vector to construct recombinant of plasmid pMD19-T-PS1and pMD19-T-TS. The amplified PEDV S1gene and TGEV S gene were2367bp and1896bp in length, which respectively encoded789and632amino acids according to the sequence analysis of these two recombinant plamids. Sequence alignment analysis showed that the deduced amino acids from PEDV SC-L strain S1gene and TGEV SC-H strain S gene respectively shared88.8%-98.6%and95.5%-99.7%homology with other PEDV strains and TGEV strains. The eukaryotic plasmid pVAXD-PS1, pVAXD-TS and co-expression plasmid pVAXD-PSl-TS were constructed and tranfected into COS-7cells. Vitro expression of the recombinant plasmids were confirmed via indirect immunofluorscence assay, which indicated that the eukaryotic expression plasmids were correctly constructed and successfully displayed specific immunofluorscence after transfected into COS-7cells, demonstrating that the three contrusted plamids pVAXD-PS1, pVAXD-TS and pVAXD-PS1-TS could successfully express in vitro, these research offered a foundation on which to further develop PEDV and TGEV DNA vaccines.
4. Oral immunization of attenuated Salmonella typhimurium harbouring PEDV S and TGEV S vaccine
The eukaryotic expression plasmids pVAXD-PS1, pVAXD-TS and pVAXD-PS1-TS were transformed by electroporation into attenuated Salmonella typhimurium SL7207to construct recombinant attenuated Salmonella typhimurium harbouring double-gene of PEDV and TGEV. After orally inoculated with a recombinant attenuated Salmonella on mice, we extract total cellular RNA from the terminal ileum of inoculated mice and detected the transcription of S1and S gene via RT-PCR. In safety analysis, the mice were orally inoculated with recombinant Salmonella at the dosage of1×109CFU per unit, the results showed that recombinant strain was relatively safe. Mice were orally inoculated at dosages of1×109CFU per unit of mixed recombinant strains SL7207(pVAXD-PS1) and SL7207(pVAXD-TS), and SL7207(pVAXD-PS1-TS) at the dosage of1×109CFU per unit, specific serum IgG and intestinal mucosal IgA antibody were detected by indirect ELISA. The results indicated that the recombinant attenuated Salmonella can provide good immunogenicity by effectively eliciting production of specified PEDV and TGEV serum IgG and intestinal IgA antibodies. Comparing the antibody levels between single-gene and double-gene immunity group, highest level of specific serum IgG and intestinal mucosal IgA antibody comes from single-gene immunity group. And was still significantly higher than (P<0.05) other two immunity group6weeks post-immunization. This study laid a foundation for the research and application of PEDV and TGEV oral DNA vaccine.
1.猪流行性腹泻病毒的分离鉴定
本文用Vero细胞成功地从仔猪腹泻粪样中分离了PEDV四川分离株(SC-L株),并对其生物学特性进行了初步研究。PEDV SC-L在Vero细胞上连续传代,增殖稳定,典型病变在接毒后48小时开始出现,表现为细胞变圆、细胞肿胀、最终细胞皱缩、破碎并脱落。通过透射电镜观察发现：病毒粒子在感染细胞胞浆内可见散在或呈晶格状排列的空心或实心的病毒粒子；病毒粒子成堆或成串排列在细胞膜上,与细胞膜融合,然后以胞外分泌的方式被释放到细胞膜外。病毒对5-溴脱氧尿核苷不敏感,是一种RNA病毒,对氯仿、乙醚敏感,60℃或以上处理30min便会失去感染力。通过对其S基因S1区进行序列测定,发现与韩国DR13株同源性最高,核苷酸同源性高达99.2%。遗传进化树分析显示所分离毒株与韩国DR13分离株亲缘关系最近。该毒株的成功分离为该病的诊断和防制积累了基础材料。
2. PEDV S基因主要抗原区域基因片断的克隆及原核表达研究
采用PCR方法从PEDV S基因TA克隆质粒中扩增出S基因的主要抗原区域基因片段Sa,扩增得到的Sa片段插入到pMD19-T simple中,构建的重组质粒命名为pMD19-T-Sa。对核苷酸序列进行测定和分析：扩增的基因长度为739bp,可编码247个氨基酸残基。本研究将Sa插入pET-32a(+)原核表达质粒,构建了原核表达质粒pET-32a(+)-PEDV-Sa,在大肠杆菌BL21中进行原核表达,SDS-PAGE电泳鉴定,原核表达质粒pET-32a(+)-PEDV-Sa表达的目的融合蛋白大小为45Kd,表达的蛋白经亲和层析法纯化后免疫家兔,制备PEDV Sa重组蛋白的多克隆抗血清。
3. PEDV S基因与TGEV S基因双基因共表达真核质粒的构建
采用RT-PCR扩增PEDV S基因S1区(包含病毒的主要中和表位和受体结合区域)和TGEV SC-H株S基因(包含A、B、C、D四个抗原位点),分别将其插入pMD19-T simple载体,构建重组质粒pMD19-T-PSl与pMD19-T-TS。对pMD19-T-PS1与pMD19-T-TS的核苷酸序列进行测定：克隆的PEDV SC-L株S1基因长约2367bp,编码789个氨基酸残基,TGEV SC-H株S基因长约1896bp,编码632个氨基酸残基。序列比对分析显示：PEDV SC-L株S1基因编码的氨基酸序列与其它PEDV毒株的同源性在88.8%-98.6%,TGEV SC-H株与其它TGEV毒株S基因编码的氨基酸序列同源性在95.5%～99.7%之间。研究以pVAXD双启动子真核表达质粒为载体,构建了能够同时表达PEDV S1和TGEV S基因的真核表达质粒pVAXD-PS1-TS以及分别单独表达PEDV S1基因和TGEV S基因的真核表达质粒pVAXD-PS1和pVAXD-TS。将构建成功的三种真核表达质粒以脂质体法分别转染COS-7细胞,通过间接免疫荧光检测发现：真核表达质粒转染的COS-7细胞在倒置荧光显微镜下呈现特异性荧光；表明上述构建的三种重组质粒可在体外表达。PEDV S1基因与TGEV S基因真核表达质粒pV AXD-PS1-TS、pV AXD-PS1与pVAXD-TS的成功构建为PEDV和TGEV DNA疫苗的进一步研究奠定了基础。
4.携带PEDV S和TGEV S双基因的减毒沙门氏菌口服疫苗免疫研究
研究将三种真核表达质粒pV AXD-PS1-TS、pV AXD-PS1与pVAXD-TS分别电转化入减毒鼠伤寒沙门氏菌SL7207中,构建了同时携带PEDV S1和TGEV S基因的重组减毒沙门氏菌SL7207(pVAXD-PS1-TS);以及携带PEDV S1基因的重组减毒沙门氏菌SL7207(pVAXD-PS1)和携带TGEV S基因的重组减毒沙门氏菌SL7207(pVAXD-TS)。小鼠灌胃接种重组减毒沙门氏菌后,提取小鼠肠道细胞总RNA,通过RT-PCR检测S1与S基因的转录。将重组菌SL7207(pVAXD-PS1)与SL7207(pVAXD-TS)混合以1×109CFU/只的剂量灌胃接种小鼠,SL7207(pVAXD-PS1-TS)以1×109CFU/只的剂量灌胃接种,间接ELISA方法检测免疫后小鼠的特异性PEDV和TGEV血清IgG以及肠道IgA抗体水平。结果：重组减毒沙门氏菌可有效激发免疫小鼠特异性PEDV(?)TGEV血清IgG以及肠道IgA抗体的产生,具有良好的免疫原性。通过比较单基因混合免疫组和双基因免疫组之间的抗体水平进一步表明：单基因混合免疫组的特异性血清IgG与肠道IgA抗体在三个免疫组中最高,且在免疫后第六周显著高于(P<0.05)其它免疫组。研究为PEDV和TGEV口服DNA疫苗的研究和应用奠定了基础。
Porcine Epidemic Diarrhea Virus (PEDV) and Transmissible Gastroenteritis Virus (TGEV) are major factor of swine viral diarrhea, and diarrhea of newborn piglets less than2weeks old caused by these two pathogens is particularly serious, which leads to severe vomiting, diarrhea and dehydration on infected piglets and nearly100%mortality rate, resulting serious economic losses for the swine industry. Thus, the enhancement of swine's specific enteric mucosal immunity plays a vital role in the prevention and therapy for both PEDV and TGEV. Based on the clinically isolated and then identified PEDV, the shared pathogenic mechanism and immune characteristics of PEDV and TGEV are taken into consideration to study on the the pathogenic mechanism of the innovatively contrusted oral DNA vaccine that could enhance mucosal immunity efficiency:PEDV S gene and TGEV S gene are used as target genes and inserted into dual promoter of eukaryotic expression vector pVAXD to conduct and study the immunogenicity of PEDV and TGEV bivalent DNA vaccine delivered by attenuated Salmonella typhimurium SL7207. The main contents were as follows:
1. Isolation and identification of Porcine Epidemic Diarrhea Virus (PEDV)
In this study, PEDV Sichuan strain SC-L was successfully isolated from diarrhea fecal samples via Vero cells, which followed by preliminary study on its biological characteristics. The proliferation of PEDV SC-L on Vero cells was stable during serial passages and typical lesions appeares48hours post-inoculation, the cells get round, swell, and ultimately shrink, splid, and shed from the culture plate. Observed by transmission electron microscopy, the hollow or solid virus particles are scattered or in lattice-like arrangement in infected intracytoplasm; the virus particles pile or cluster on the cell membrane, fusing with the cell membrane and secreting outside the cell membrane. This RNA virus is insensitive to5-bromodeoxyuridine urinary nucleosides but sensitive to chloroform and aether, it loses infectbility when in the circumstance of60℃or above for 30min. By Sequencing S1domain of S gene, it is in the highest homology with South Korea DR13strains with nucleotide homology high up to99.2%. The phylogenetic tree analysis shows that the isolated strains and South Korea DR13isolated strains are in the closest genetic relationship. This successfully isolated strain accounts for material foundation of diagnosis and prevention for PEDV.
2. Cloning and prokaryotic expression of PEDV S gene main antigtic domain fragment
The PEDV S gene main antigtic domain fragment Sa were amplified from the PEDV S gene TA cloning plasmid via PCR and then cloned into the pMD19-T simple vector to construct the recombinant plasmid pMD19-T-Sa. The amplified gene is739bp in length and is able to encode247amino acids accroding to the sequence analysis. The recombinant prokaryotic plasmid pET-32a(+)-PEDV-Sa with Sa instered into pET-32a(+) prokaryotic expression plasmid could produce45Kd fusion protein in E.coli BL21, which is detected by SDS-PAGE electrophoresis. The expressed protein was purified by affinity chromatography immune and polyclonal antiserum was prepared by immunizing rabbits with this PEDV Sa recombinant protein.
3. The construction of PEDV S gene and TGEV S gene co-expression eukaryotic plasmid
The RT-PCR amplified S1region of PEDV S gene that contains main neutralization epitope and receptor binding region of the virus, and5'fragment of S gene of the TGVE SC-H strain that covers A and B, and C, and D antigens points were respectively cloned into pMD-19T simple vector to construct recombinant of plasmid pMD19-T-PS1and pMD19-T-TS. The amplified PEDV S1gene and TGEV S gene were2367bp and1896bp in length, which respectively encoded789and632amino acids according to the sequence analysis of these two recombinant plamids. Sequence alignment analysis showed that the deduced amino acids from PEDV SC-L strain S1gene and TGEV SC-H strain S gene respectively shared88.8%-98.6%and95.5%-99.7%homology with other PEDV strains and TGEV strains. The eukaryotic plasmid pVAXD-PS1, pVAXD-TS and co-expression plasmid pVAXD-PSl-TS were constructed and tranfected into COS-7cells. Vitro expression of the recombinant plasmids were confirmed via indirect immunofluorscence assay, which indicated that the eukaryotic expression plasmids were correctly constructed and successfully displayed specific immunofluorscence after transfected into COS-7cells, demonstrating that the three contrusted plamids pVAXD-PS1, pVAXD-TS and pVAXD-PS1-TS could successfully express in vitro, these research offered a foundation on which to further develop PEDV and TGEV DNA vaccines.
4. Oral immunization of attenuated Salmonella typhimurium harbouring PEDV S and TGEV S vaccine
The eukaryotic expression plasmids pVAXD-PS1, pVAXD-TS and pVAXD-PS1-TS were transformed by electroporation into attenuated Salmonella typhimurium SL7207to construct recombinant attenuated Salmonella typhimurium harbouring double-gene of PEDV and TGEV. After orally inoculated with a recombinant attenuated Salmonella on mice, we extract total cellular RNA from the terminal ileum of inoculated mice and detected the transcription of S1and S gene via RT-PCR. In safety analysis, the mice were orally inoculated with recombinant Salmonella at the dosage of1×109CFU per unit, the results showed that recombinant strain was relatively safe. Mice were orally inoculated at dosages of1×109CFU per unit of mixed recombinant strains SL7207(pVAXD-PS1) and SL7207(pVAXD-TS), and SL7207(pVAXD-PS1-TS) at the dosage of1×109CFU per unit, specific serum IgG and intestinal mucosal IgA antibody were detected by indirect ELISA. The results indicated that the recombinant attenuated Salmonella can provide good immunogenicity by effectively eliciting production of specified PEDV and TGEV serum IgG and intestinal IgA antibodies. Comparing the antibody levels between single-gene and double-gene immunity group, highest level of specific serum IgG and intestinal mucosal IgA antibody comes from single-gene immunity group. And was still significantly higher than (P<0.05) other two immunity group6weeks post-immunization. This study laid a foundation for the research and application of PEDV and TGEV oral DNA vaccine.
引文
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