NDV HN基因和ILTV gD基因重組腺病毒的構(gòu)建及免疫效果評價(jià)
發(fā)布時(shí)間：2018-08-19 21:17
【摘要】：新城疫(Newcastle disease,ND)是由新城疫病毒(Newcastle disease virus,NDV)引起的一種急性、高度接觸性傳染性疾病,在全世界范圍內(nèi)流行傳播,屬于嚴(yán)重危害養(yǎng)雞業(yè)的疫病之一。血凝素-神經(jīng)氨酸酶(H-N)蛋白具有良好的免疫原性,已成為目前研究新城疫基因工程疫苗的首選目的基因之一。雞傳染性喉氣管炎(Infectious laryngotracheitis,ILT)是由傳染性喉氣管炎病毒(Infectious laryngotracheitis virus,ILTV)引起的一種傳染病。gD糖蛋白是ILTV復(fù)制和誘導(dǎo)宿主產(chǎn)生免疫應(yīng)答反應(yīng)中的主要保護(hù)性抗原,該蛋白編碼基因高度保守,為ILTV吸附和侵染所必須的,可以誘導(dǎo)機(jī)體產(chǎn)生體液和細(xì)胞免疫應(yīng)答,由其引起的細(xì)胞免疫應(yīng)答效應(yīng)高于gB和gC基因。目前,市面上應(yīng)用較多是NDV和ILTV弱毒疫苗,弱毒疫苗有返毒返強(qiáng)等缺點(diǎn),而基因工程疫苗安全性高,還可構(gòu)建多價(jià)疫苗和多聯(lián)苗,在新型疫苗研究中有開闊的前景。1.HN和gD基因重組腺病毒的構(gòu)建與鑒定試驗(yàn)通過RT-PCR/PCR方法擴(kuò)增得到HN和gD基因;將目的基因通過T-A克隆至pMD19-T載體上,獲得質(zhì)粒pMD-HN和pMD-gD;經(jīng)酶切及測序鑒定后與穿梭載體pDC_(315)-EGFP連接,分別獲得重組腺病毒穿梭質(zhì)粒pDC_(315)-HN-EGFP、pDC_(315)-gD-EGFP和pDC_(315)-HN-gD-EGFP;將重組腺病毒穿梭質(zhì)粒與腺病毒大骨架共轉(zhuǎn)染至HEK293細(xì)胞;經(jīng)RT-PCR檢測,構(gòu)建的重組腺病毒可以感染HEK293細(xì)胞;Western blotting檢測HN蛋白和gD蛋白在HE293細(xì)胞中高表達(dá);擴(kuò)增純化后的重組腺病毒經(jīng)半數(shù)組織感染劑量法(TICD_(50))測定其病毒滴度,結(jié)果顯示構(gòu)建的重組腺病毒pBH-HN-EGFP、pBH-gD-EGFP、pBH-HN-gD-EGFP和pBH-EGFP的滴度分別為10~(10.5)TCID_(50)/mL、107.875TCID_(50)/mL、10~(10.5) TCID_(50)/mL和1011.25 TCID_(50)/mL,符合后續(xù)動物試驗(yàn)所需的病毒滴度。2.ND組,HN和gD基因重組腺病毒的免疫效果評價(jià)將PBS組、pBH-EGFP、NDV弱毒疫苗(Lasota)組、重組腺病毒pBH-HN-EGFP組和pBH-HN-gD-EGFP組進(jìn)行免疫試驗(yàn)。雞增重試驗(yàn)結(jié)果表明,重組腺病毒疫苗對SPF雞的生長無明顯抑制作用;免疫后抗體水平結(jié)果顯示:NDV弱毒疫苗(Lasota)組與重組腺病毒pBH-HN-EGFP組和pBH-HN-gD-EGFP組差異不顯著(p0.05),均與PBS組和pBH-EGFP組差異顯著(p0.05);淋巴細(xì)胞檢測結(jié)果表明:NDV弱毒疫苗(Lasota)組與重組腺病毒組均能誘導(dǎo)CD3~+、CD4~+、CD8~+和Bu-1a~+細(xì)胞增殖。說明該重組腺病毒可以誘導(dǎo)雞產(chǎn)生體液免疫和細(xì)胞免疫。3.ILT組,HN和gD基因重組腺病毒的免疫效果評價(jià)將PBS組、pBH-EGFP、ILTV弱毒疫苗K317株組、重組腺病毒pBH-gD-EGFP組和pBH-HN-gD-EGFP組進(jìn)行免疫試驗(yàn)。雞增重試驗(yàn)結(jié)果表明:重組腺病毒疫苗對SPF雞的生長無明顯抑制作用;免疫后抗體檢測結(jié)果顯示:ILTV弱毒疫苗K317株組與重組腺病毒pBH-gD-EGFP組和pBH-HN-gD-EGFP組差異不顯著(p0.05),均與PBS組和pBH-EGFP組差異顯著(p0.05);淋巴細(xì)胞檢測結(jié)果表明:ILTV弱毒疫苗K317株組與重組腺病毒組均能誘導(dǎo)CD3~+、CD4~+、CD8~+和Bu-1a~+細(xì)胞增殖。說明該重組腺病毒可以誘導(dǎo)機(jī)體產(chǎn)生體液免疫和細(xì)胞免疫。綜上所述,本研究成功構(gòu)建了重組腺病毒pBH-HN-gD-EGFP活載體疫苗,且能使機(jī)體產(chǎn)生體液免疫和細(xì)胞免疫,可以作為ND和ILT的新型候選疫苗。
[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.
【學(xué)位授予單位】：天津農(nóng)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S852.4
本文編號：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.
【學(xué)位授予單位】：天津農(nóng)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S852.4
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