Asia I型、O型口蹄疫复合多表位重组酵母菌的构建与实验免疫
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摘要
口蹄疫(Foot-and-Mouth Disease,FMD)是由口蹄疫病毒(Foot-and-Mouth Disease Virus,FMDV)引起的主要侵害偶蹄动物感染的一种急性、热性、高度接触性传染病。口蹄疫病毒属小核糖核酸病毒科(Picornaviridae),口蹄疫病毒属共有7个血清型,80多个亚型。由于口蹄疫病毒血清型多、血清型之间无交叉免疫现象,故给口蹄疫的防制带来了很大的困难。而目前所用的口蹄疫常规灭活疫苗和紧急疫苗均是由活病毒添加灭活剂而制成的,存在着由于活病毒逃逸和灭活不彻底引起新疫情爆发的危险。本研究选用口蹄疫病毒的主要抗原位点VP1蛋白和顺式裂解元件2A作为主体。选用含有FMDV不同毒株的VP1上多个抗原表位的T、B细胞表位基因组成的Epi基因盒,加上Th2表位和霍乱毒素B亚单位(CTB),形成多表位片段CTB-Th2-Epi(简称CTE)作为辅助抗原,从而增强免疫,为进一步研制FMD的基因工程疫苗提供一种新的思路。
本研究首先应用PCR方法扩增出FMDV Asia I/JL/05株和FMDV O/NY/00株VP1-2A基因,然后对PCR扩增产物进行纯化回收,并将其分别克隆到pMD18-T载体上。经测序后,与已知序列进行比对分析。应用限制性内切酶消化pMD18-T-CTE(Asia I型)、pMD18-T-CTE(O型)获得多表位CTE片段。并将两段基因进一步克隆到pPIC9K表达载体上,并转化进酵母菌GS115内,经甲醇诱导后,通过SDS-PAGE鉴定目的蛋白的表达情况,Western-Blot检测重组蛋白的活性。并应用纯化后的蛋白免疫小鼠和豚鼠,通过间接ELISA、MTT和ELISPOT、细胞因子含量来评价重组蛋白的免疫效果。
结果成功地扩增出FMDV Asia I型、O型VP1-2A基因。重组克隆pMD18-T-VP1-2A(Asia I型)、pMD18-T-VP1-2A(O型)经测序后,与Asia I/JL/05株和FMDV O/NY/00株的核苷酸一致性分别为100%和99.72%。成功构建了pPIC9K-VP1-2A-CTE ( Asia I型)和pPIC9K-VP1-2A-CTE(O型)重组表达质粒,并转化进GSl15酵母菌,经SDS-PAGE鉴定后蛋白已成功表达,表达量分别为75 mg/L和80 mg/L,经Western blot鉴定表达的蛋白具有抗原性。免疫小鼠后,经间接ELISA检测,各免疫组均可产生特异性抗体,并在二免后第三周达到最高峰;经MTT、ELISPOT和细胞因子检测表明,两个重组蛋白免疫组均表现出相应的细胞免疫应答,且均高于PBS对照组和GS115/pPIC9K对照组,但低于灭活苗免疫组。免疫豚鼠后,经间接ELISA检测,各免疫组均可产生特异性抗体,并在二免后达到最高峰;经MTT和细胞因子检测表明,豚鼠细胞免疫应答水平比小鼠的细胞免疫应答水平增强,但低于灭活苗免疫组。
综上所述,以Asia I型、O型口蹄疫病毒VP1-2A为主体,以复合多表位片段CTE为辅助抗原在酵母菌GSl15所表达的重组蛋白具有较好的免疫原性,可以作为口蹄疫防制的候选疫苗应用于控制口蹄疫的传播。
Foot-and-mouth disease (FMD) caused by foot and mouth disease virus (FMDV) is a highly contagious and economically devastating disease, which always inflict cloven-hoofed animals. FMDV is the prototype member of the Aphthovirus genus of the family Picornaviridae. There are 7 serotypes and more than 80 subtypes of FMDV, so it brought great difficulties to control it. Because of following risky factors such as the incomplete inactivation of the virus and escape of live virus, new type of vaccine which should be safer and more effective are under intensive study worldwide. In this paper we selected VP1 gene of serotype Asia I and O of FMDV and non-strcture protein 2A gene as the major antigen , and other epitopes gene which composed of the cholera toxin B subunit gene, Th2 cell epitopes and the box constructed with T cell andB cell epitope gene.
We amplified VP1-2A gene of Asia I/JL/05 and O/NY/00 of FMDV by PCR .Then, VP1-2A gene of Asia I/JL/05 and O/NY/00 of FMDV was ligated into pMD18-T vector. The recombinant clone, pMD18-T-VP1-2A, were sequenced. The nucleotide sequence of VP1-2A was compared and analyzed with the Asia I/JL/05 and O/NY/00 of FMDV. Then we obtained CTE multiple epitope fragment by restricted digestion of pMD18-T-CTE plasmid of type Asia I and O FMDV. Further the VP1-2A and CTE fragment was ligated into pPIC9K expression vector. Then, it was transformed into Pichia pastoris GS115 and was induced with methanol to express recombinant VP1-2A and CTE fusion protein. The expression of target protein was detected by SDS-PAGE and Western blot assay. Mice and guinea pigs were inoculated with these recombinant vaccines. And the FMDV specific antibody, T lymphoproliferation and T lymphocyte subpopulation were detected.
The results showed that VP1-2A gene of serotype Asia I and O of FMDV was successfully amplified and cloned. Sequencing results showed that the VP1-2A gene of Asia I of FMDV shares 100% homology in nucleotide sequence, and the VP1-2A gene of O of FMDV shares 99.7% homology in nucleotide sequence. Recombinant expression plasmid pPIC9K-VP1-2A-CTE was constructed by inserting of serotype Asia I and O of FMDV VP1-2A and CTE gene into yeast expression vector pPIC9K. Secondly, plasmid pPIC9K-VP1-2A-CTE of serotype Asia I and O of FMDV was transformed into GS115 cells by electroporation.SDS-PAGE results showed that expression product could be secreted into media and existed in dimer form, production of target protein could reach 75 mg and 80 mg per litter.And the Western blot results showed that expression product could be detected by FMDV antiserum, which proved the good antigen specificity of expressed protein. Mice and guinea pigs were inoculated intramuscularly with recombinant vaccines and the inactivated vaccine. The FMDV specific antibody, T lymphoproliferation and cytokine concentration results showed that in all recombinant vaccines inoculated groups, could be induced specific humoral and cellular immune responses. In mice, the inactivated vaccine had the highest immunogenicity. Comparing with GS115/pPIC9K group and PBS group the GS115/pPIC9K-VP1-2A-CTE of serotype Asia I and O of FMDV group could induce not only approximately same FMDV antibody level, but also higher cellular immune responses. In guinea pigs, the GS115/pPIC9K-VP1-2A-CTE of serotype Asia I and O of FMDV group had higher cellular immune responses than in mice.but only had weaker humoral responses than inactivated vaccine.
In conclusion, we selected VP1 gene of serotype Asia I and O of FMDV and non-strcture protein 2A gene as the major antigen , and CTE multi-epitopes fagment as the second antigen, and expressed them in the expression system of Pichia. The protein has good immunogenicity.It could be as the candidate vaccines to contol foot and mouth disease.
本研究首先应用PCR方法扩增出FMDV Asia I/JL/05株和FMDV O/NY/00株VP1-2A基因,然后对PCR扩增产物进行纯化回收,并将其分别克隆到pMD18-T载体上。经测序后,与已知序列进行比对分析。应用限制性内切酶消化pMD18-T-CTE(Asia I型)、pMD18-T-CTE(O型)获得多表位CTE片段。并将两段基因进一步克隆到pPIC9K表达载体上,并转化进酵母菌GS115内,经甲醇诱导后,通过SDS-PAGE鉴定目的蛋白的表达情况,Western-Blot检测重组蛋白的活性。并应用纯化后的蛋白免疫小鼠和豚鼠,通过间接ELISA、MTT和ELISPOT、细胞因子含量来评价重组蛋白的免疫效果。
结果成功地扩增出FMDV Asia I型、O型VP1-2A基因。重组克隆pMD18-T-VP1-2A(Asia I型)、pMD18-T-VP1-2A(O型)经测序后,与Asia I/JL/05株和FMDV O/NY/00株的核苷酸一致性分别为100%和99.72%。成功构建了pPIC9K-VP1-2A-CTE ( Asia I型)和pPIC9K-VP1-2A-CTE(O型)重组表达质粒,并转化进GSl15酵母菌,经SDS-PAGE鉴定后蛋白已成功表达,表达量分别为75 mg/L和80 mg/L,经Western blot鉴定表达的蛋白具有抗原性。免疫小鼠后,经间接ELISA检测,各免疫组均可产生特异性抗体,并在二免后第三周达到最高峰;经MTT、ELISPOT和细胞因子检测表明,两个重组蛋白免疫组均表现出相应的细胞免疫应答,且均高于PBS对照组和GS115/pPIC9K对照组,但低于灭活苗免疫组。免疫豚鼠后,经间接ELISA检测,各免疫组均可产生特异性抗体,并在二免后达到最高峰;经MTT和细胞因子检测表明,豚鼠细胞免疫应答水平比小鼠的细胞免疫应答水平增强,但低于灭活苗免疫组。
综上所述,以Asia I型、O型口蹄疫病毒VP1-2A为主体,以复合多表位片段CTE为辅助抗原在酵母菌GSl15所表达的重组蛋白具有较好的免疫原性,可以作为口蹄疫防制的候选疫苗应用于控制口蹄疫的传播。
Foot-and-mouth disease (FMD) caused by foot and mouth disease virus (FMDV) is a highly contagious and economically devastating disease, which always inflict cloven-hoofed animals. FMDV is the prototype member of the Aphthovirus genus of the family Picornaviridae. There are 7 serotypes and more than 80 subtypes of FMDV, so it brought great difficulties to control it. Because of following risky factors such as the incomplete inactivation of the virus and escape of live virus, new type of vaccine which should be safer and more effective are under intensive study worldwide. In this paper we selected VP1 gene of serotype Asia I and O of FMDV and non-strcture protein 2A gene as the major antigen , and other epitopes gene which composed of the cholera toxin B subunit gene, Th2 cell epitopes and the box constructed with T cell andB cell epitope gene.
We amplified VP1-2A gene of Asia I/JL/05 and O/NY/00 of FMDV by PCR .Then, VP1-2A gene of Asia I/JL/05 and O/NY/00 of FMDV was ligated into pMD18-T vector. The recombinant clone, pMD18-T-VP1-2A, were sequenced. The nucleotide sequence of VP1-2A was compared and analyzed with the Asia I/JL/05 and O/NY/00 of FMDV. Then we obtained CTE multiple epitope fragment by restricted digestion of pMD18-T-CTE plasmid of type Asia I and O FMDV. Further the VP1-2A and CTE fragment was ligated into pPIC9K expression vector. Then, it was transformed into Pichia pastoris GS115 and was induced with methanol to express recombinant VP1-2A and CTE fusion protein. The expression of target protein was detected by SDS-PAGE and Western blot assay. Mice and guinea pigs were inoculated with these recombinant vaccines. And the FMDV specific antibody, T lymphoproliferation and T lymphocyte subpopulation were detected.
The results showed that VP1-2A gene of serotype Asia I and O of FMDV was successfully amplified and cloned. Sequencing results showed that the VP1-2A gene of Asia I of FMDV shares 100% homology in nucleotide sequence, and the VP1-2A gene of O of FMDV shares 99.7% homology in nucleotide sequence. Recombinant expression plasmid pPIC9K-VP1-2A-CTE was constructed by inserting of serotype Asia I and O of FMDV VP1-2A and CTE gene into yeast expression vector pPIC9K. Secondly, plasmid pPIC9K-VP1-2A-CTE of serotype Asia I and O of FMDV was transformed into GS115 cells by electroporation.SDS-PAGE results showed that expression product could be secreted into media and existed in dimer form, production of target protein could reach 75 mg and 80 mg per litter.And the Western blot results showed that expression product could be detected by FMDV antiserum, which proved the good antigen specificity of expressed protein. Mice and guinea pigs were inoculated intramuscularly with recombinant vaccines and the inactivated vaccine. The FMDV specific antibody, T lymphoproliferation and cytokine concentration results showed that in all recombinant vaccines inoculated groups, could be induced specific humoral and cellular immune responses. In mice, the inactivated vaccine had the highest immunogenicity. Comparing with GS115/pPIC9K group and PBS group the GS115/pPIC9K-VP1-2A-CTE of serotype Asia I and O of FMDV group could induce not only approximately same FMDV antibody level, but also higher cellular immune responses. In guinea pigs, the GS115/pPIC9K-VP1-2A-CTE of serotype Asia I and O of FMDV group had higher cellular immune responses than in mice.but only had weaker humoral responses than inactivated vaccine.
In conclusion, we selected VP1 gene of serotype Asia I and O of FMDV and non-strcture protein 2A gene as the major antigen , and CTE multi-epitopes fagment as the second antigen, and expressed them in the expression system of Pichia. The protein has good immunogenicity.It could be as the candidate vaccines to contol foot and mouth disease.
引文
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