NDV HN基因和ILTV gD基因重组腺病毒的构建及免疫效果评价
发布时间：2018-08-19 21:17
【摘要】：新城疫(Newcastle disease,ND)是由新城疫病毒(Newcastle disease virus,NDV)引起的一种急性、高度接触性传染性疾病,在全世界范围内流行传播,属于严重危害养鸡业的疫病之一。血凝素-神经氨酸酶(H-N)蛋白具有良好的免疫原性,已成为目前研究新城疫基因工程疫苗的首选目的基因之一。鸡传染性喉气管炎(Infectious laryngotracheitis,ILT)是由传染性喉气管炎病毒(Infectious laryngotracheitis virus,ILTV)引起的一种传染病。gD糖蛋白是ILTV复制和诱导宿主产生免疫应答反应中的主要保护性抗原,该蛋白编码基因高度保守,为ILTV吸附和侵染所必须的,可以诱导机体产生体液和细胞免疫应答,由其引起的细胞免疫应答效应高于gB和gC基因。目前,市面上应用较多是NDV和ILTV弱毒疫苗,弱毒疫苗有返毒返强等缺点,而基因工程疫苗安全性高,还可构建多价疫苗和多联苗,在新型疫苗研究中有开阔的前景。1.HN和gD基因重组腺病毒的构建与鉴定试验通过RT-PCR/PCR方法扩增得到HN和gD基因;将目的基因通过T-A克隆至pMD19-T载体上,获得质粒pMD-HN和pMD-gD;经酶切及测序鉴定后与穿梭载体pDC_(315)-EGFP连接,分别获得重组腺病毒穿梭质粒pDC_(315)-HN-EGFP、pDC_(315)-gD-EGFP和pDC_(315)-HN-gD-EGFP;将重组腺病毒穿梭质粒与腺病毒大骨架共转染至HEK293细胞;经RT-PCR检测,构建的重组腺病毒可以感染HEK293细胞;Western blotting检测HN蛋白和gD蛋白在HE293细胞中高表达;扩增纯化后的重组腺病毒经半数组织感染剂量法(TICD_(50))测定其病毒滴度,结果显示构建的重组腺病毒pBH-HN-EGFP、pBH-gD-EGFP、pBH-HN-gD-EGFP和pBH-EGFP的滴度分别为10~(10.5)TCID_(50)/mL、107.875TCID_(50)/mL、10~(10.5) TCID_(50)/mL和1011.25 TCID_(50)/mL,符合后续动物试验所需的病毒滴度。2.ND组,HN和gD基因重组腺病毒的免疫效果评价将PBS组、pBH-EGFP、NDV弱毒疫苗(Lasota)组、重组腺病毒pBH-HN-EGFP组和pBH-HN-gD-EGFP组进行免疫试验。鸡增重试验结果表明,重组腺病毒疫苗对SPF鸡的生长无明显抑制作用;免疫后抗体水平结果显示:NDV弱毒疫苗(Lasota)组与重组腺病毒pBH-HN-EGFP组和pBH-HN-gD-EGFP组差异不显著(p0.05),均与PBS组和pBH-EGFP组差异显著(p0.05);淋巴细胞检测结果表明:NDV弱毒疫苗(Lasota)组与重组腺病毒组均能诱导CD3~+、CD4~+、CD8~+和Bu-1a~+细胞增殖。说明该重组腺病毒可以诱导鸡产生体液免疫和细胞免疫。3.ILT组,HN和gD基因重组腺病毒的免疫效果评价将PBS组、pBH-EGFP、ILTV弱毒疫苗K317株组、重组腺病毒pBH-gD-EGFP组和pBH-HN-gD-EGFP组进行免疫试验。鸡增重试验结果表明:重组腺病毒疫苗对SPF鸡的生长无明显抑制作用;免疫后抗体检测结果显示:ILTV弱毒疫苗K317株组与重组腺病毒pBH-gD-EGFP组和pBH-HN-gD-EGFP组差异不显著(p0.05),均与PBS组和pBH-EGFP组差异显著(p0.05);淋巴细胞检测结果表明:ILTV弱毒疫苗K317株组与重组腺病毒组均能诱导CD3~+、CD4~+、CD8~+和Bu-1a~+细胞增殖。说明该重组腺病毒可以诱导机体产生体液免疫和细胞免疫。综上所述,本研究成功构建了重组腺病毒pBH-HN-gD-EGFP活载体疫苗,且能使机体产生体液免疫和细胞免疫,可以作为ND和ILT的新型候选疫苗。
[Abstract]:Newcastle disease (ND) is an acute, highly contagious disease caused by Newcastle disease virus (NDV), which spreads all over the world. It is one of the most serious diseases that endanger chicken industry. Infectious laryngotracheitis (ILT) is an infectious disease caused by infectious laryngotracheitis virus (ILTV). GD glycoprotein is a major protective antigen in the replication and induction of immune response by ILTV. The gene encoding the protein is highly conserved, which is necessary for the adsorption and infection of ILTV. It can induce humoral and cellular immune responses. The cellular immune responses induced by the protein are higher than those of gB and gC genes. At present, the attenuated NDV and ILTV vaccines are widely used in the market, and the attenuated vaccines have the disadvantages of reverting to virulence, and the genetically engineered vaccines are safe. The recombinant adenovirus containing HN and gD genes was amplified by RT-PCR/PCR, and the target genes were cloned into pMD19-T vector by T-A to obtain plasmids pMD-HN and pMD-gD, which were identified by enzyme digestion and sequencing. The recombinant adenovirus shuttle plasmids pDC_ (315) - HN-EGFP, pDC_ (315) - gD-EGFP and pDC_ (315) - HN-gD-EGFP were obtained by connecting the shuttle vectors pDC_ (315) - EGFP. The recombinant adenovirus shuttle plasmids were co-transfected into HEK293 cells with adenovirus macroskeleton. The recombinant adenovirus could infect HEK293 cells by RT-PCR. The HN eggs were detected by Western blotting. The recombinant adenovirus was highly expressed in HE293 cells, and the titers of pBH-HN-EGFP, pBH-gD-EGFP, pBH-HN-gD-EGFP and pBH-EGFP were 10~ (10.5) TCID_ (50)/mL, 107.875TCID_ (50)/mL, 10~ (10.5) TCID_ (5) respectively. 0) / mL and 1011.25 TCID_ (50) / mL, which accorded with the virus titers required for subsequent animal tests. 2. ND group, HN and gD gene recombinant adenovirus immune efficacy evaluation will be PBS group, pBH-EGFP, NDV attenuated vaccine (Lasota) group, recombinant adenovirus pBH-HN-EGFP group and pBH-HN-gD-EGFP group immune test. Chicken weight gain test showed that recombinant adenovirus vaccine against S-HN-gD-EGFP group. The growth of PF chickens was not significantly inhibited; the antibody level after immunization showed that there was no significant difference between NDV attenuated vaccine (Lasota) group and recombinant adenovirus pBH-HN-EGFP group and pBH-HN-gD-EGFP group (p0.05), and there was significant difference between NDV attenuated vaccine (Lasota) group and recombinant adenovirus group (pBH-HN-gD-EGFP). The recombinant adenovirus could induce humoral and cellular immunity in chickens. 3. The immune efficacy of recombinant adenovirus containing HN and gD genes was evaluated in PBS group, pBH-EGFP, ILTV attenuated vaccine K317 group, recombinant adenovirus pBH-gD-EGFP group and pBH-HN-gD-EGFP group. The results of weight gain test showed that the recombinant adenovirus vaccine had no obvious inhibitory effect on the growth of SPF chickens; the results of antibody test after immunization showed that there was no significant difference between the ILTV attenuated vaccine K317 strain group and the recombinant adenovirus pBH-gD-EGFP group and the pBH-HN-gD-EGFP group (p0.05), and there was significant difference between the PBS group and the pBH-EGFP group (p0.05). LTV attenuated vaccine K317 strain group and recombinant adenovirus group could induce the proliferation of CD3~+, CD4~+, CD8~+ and Bu-1a~+ cells, which indicated that the recombinant adenovirus could induce humoral and cellular immunity. In conclusion, the recombinant adenovirus pBH-HN-gD-EGFP live vector vaccine was successfully constructed and could induce humoral and cellular immunity. Immunization can be used as a new candidate vaccine for ND and ILT.
【学位授予单位】：天津农学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.4
本文编号：2192851
[Abstract]:Newcastle disease (ND) is an acute, highly contagious disease caused by Newcastle disease virus (NDV), which spreads all over the world. It is one of the most serious diseases that endanger chicken industry. Infectious laryngotracheitis (ILT) is an infectious disease caused by infectious laryngotracheitis virus (ILTV). GD glycoprotein is a major protective antigen in the replication and induction of immune response by ILTV. The gene encoding the protein is highly conserved, which is necessary for the adsorption and infection of ILTV. It can induce humoral and cellular immune responses. The cellular immune responses induced by the protein are higher than those of gB and gC genes. At present, the attenuated NDV and ILTV vaccines are widely used in the market, and the attenuated vaccines have the disadvantages of reverting to virulence, and the genetically engineered vaccines are safe. The recombinant adenovirus containing HN and gD genes was amplified by RT-PCR/PCR, and the target genes were cloned into pMD19-T vector by T-A to obtain plasmids pMD-HN and pMD-gD, which were identified by enzyme digestion and sequencing. The recombinant adenovirus shuttle plasmids pDC_ (315) - HN-EGFP, pDC_ (315) - gD-EGFP and pDC_ (315) - HN-gD-EGFP were obtained by connecting the shuttle vectors pDC_ (315) - EGFP. The recombinant adenovirus shuttle plasmids were co-transfected into HEK293 cells with adenovirus macroskeleton. The recombinant adenovirus could infect HEK293 cells by RT-PCR. The HN eggs were detected by Western blotting. The recombinant adenovirus was highly expressed in HE293 cells, and the titers of pBH-HN-EGFP, pBH-gD-EGFP, pBH-HN-gD-EGFP and pBH-EGFP were 10~ (10.5) TCID_ (50)/mL, 107.875TCID_ (50)/mL, 10~ (10.5) TCID_ (5) respectively. 0) / mL and 1011.25 TCID_ (50) / mL, which accorded with the virus titers required for subsequent animal tests. 2. ND group, HN and gD gene recombinant adenovirus immune efficacy evaluation will be PBS group, pBH-EGFP, NDV attenuated vaccine (Lasota) group, recombinant adenovirus pBH-HN-EGFP group and pBH-HN-gD-EGFP group immune test. Chicken weight gain test showed that recombinant adenovirus vaccine against S-HN-gD-EGFP group. The growth of PF chickens was not significantly inhibited; the antibody level after immunization showed that there was no significant difference between NDV attenuated vaccine (Lasota) group and recombinant adenovirus pBH-HN-EGFP group and pBH-HN-gD-EGFP group (p0.05), and there was significant difference between NDV attenuated vaccine (Lasota) group and recombinant adenovirus group (pBH-HN-gD-EGFP). The recombinant adenovirus could induce humoral and cellular immunity in chickens. 3. The immune efficacy of recombinant adenovirus containing HN and gD genes was evaluated in PBS group, pBH-EGFP, ILTV attenuated vaccine K317 group, recombinant adenovirus pBH-gD-EGFP group and pBH-HN-gD-EGFP group. The results of weight gain test showed that the recombinant adenovirus vaccine had no obvious inhibitory effect on the growth of SPF chickens; the results of antibody test after immunization showed that there was no significant difference between the ILTV attenuated vaccine K317 strain group and the recombinant adenovirus pBH-gD-EGFP group and the pBH-HN-gD-EGFP group (p0.05), and there was significant difference between the PBS group and the pBH-EGFP group (p0.05). LTV attenuated vaccine K317 strain group and recombinant adenovirus group could induce the proliferation of CD3~+, CD4~+, CD8~+ and Bu-1a~+ cells, which indicated that the recombinant adenovirus could induce humoral and cellular immunity. In conclusion, the recombinant adenovirus pBH-HN-gD-EGFP live vector vaccine was successfully constructed and could induce humoral and cellular immunity. Immunization can be used as a new candidate vaccine for ND and ILT.
【学位授予单位】：天津农学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.4
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