表达鸡传染性喉气管炎病毒gD基因与鸡IL-2基因重组禽痘病毒的构建及其免疫效力研究
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摘要
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)引起的鸡的一种急性上呼吸道传染病,本病给养鸡业造成较大的经济损失。目前主要应用致弱的喉气管炎病毒作为弱毒疫苗,但是由于鸡传染性喉气管炎弱毒疫苗株也能引起潜伏感染,并在鸡群与鸡群之间传播过程中毒力会返强,从而成为新的感染源,因此,研究新型安全且有效的疫苗用于ILT的防制十分必要。本文将ILTV河南长葛株的gD基因与鸡IL-2基因重组禽痘病毒转移载体,得到一株重组病毒。试验研究主要从以下几个方面展开:
1.ILTV河南长葛株分离鉴定及其gB、gC和gD基因的克隆与序列分析
鸡胚尿囊膜接种和琼脂扩散试验对分离病毒进行鉴定,结果表明所分离病毒为ILTV,从而获得一株ILTV河南长葛株。并分别克隆其gB、gC和gD基因,测定其核苷酸序列,将其核苷酸与GenBank公布的ILTV相应序列进行分析和同源性比较,结果长葛分离株ILTV的gB、gC和gD基因3个序列分别与GenBank登录的ILTV gB、gC和gD基因序列存在很高的同源性,其同源性在99.7%～99.9%之间,说明ILTV的gB、gC和gD基因序列相对保守。
2.ILTV长葛株gB、gC和gD基因真核表达载体的构建及其免疫效果研究
将ILTV长葛株gB、gC和gD基因插入pcDNA3.1中,构建了真核表达载体pcDNA-gB、pcDNA-gC和pcDNA-gD。间接免疫荧光检测结果表明,三种重组质粒均能够在PK-15细胞中表达。以SPF鸡为试验动物,肌内注射构建的pcDNA-gB、pcDNA-gC、pcDNA-gD和pcDNA3.1空载体进行免疫接种。微量血清中和试验检测结果显示,构建的真核表达载体pcDNA-gB、pcDNA-gC、pcDNA-gD和活疫苗均能够诱导机体产生较高的抗体水平;MTT法检测表明构建的真核表达载体pcDNA-gB、pcDNA-gC、pcDNA-gD及活疫苗对照均能够诱导淋巴细胞增殖,同时也能够诱导强的CTL细胞的增殖。表明构建的载体pcDNA-gB、pcDNA-gC、pcDNA-gD和弱毒疫苗均能够有效诱导机体产生体液免疫和细胞免疫。
3.ILTV长葛株gD基因的表达及间接ELISA检测方法的初步建立
将ILTV gD基因插入到原核表达载体pET-28a(+)中,构建重组表达质粒pET28-gD。将pET28-gD转化到宿主表达菌BL21中,用IPTG诱导表达,SDS-PAGE结果显示表达目的蛋白大小为55ku,Western-blotting表明表达产物具有良好的免疫原性。表达的蛋白产物经纯化后作为ELISA包被抗原,初步建立了间接ELISA诊断方法,并确定了抗原最佳包被浓度为24.5μg·mL;最佳血清稀释度为1∶320,初步确定ELISA判定标准为OD_(450)≥0.25判为阳性,小于0.25为阴性。试验证明建立的间接ELISA具有很强的特异性和较高的敏感性且与多种病毒抗原无交叉反应。用初步建立的ELISA和微量血清中和试验检测临床送检的84份血清样品。结果两种方法的阳性符合率为96.5%(28/29),阴性符合率为98.2%(55/56),表明建立的方法可用于临床上ILT的检测。
4.表达ILTV长葛株gD基因重组禽痘病毒的构建及其免疫效力试验
将ILTV长葛株gD基因同源重组到禽痘病毒基因组中,得到能高效表达ILTV gD基因的重组禽痘病毒(rFPV-ILTV-gD)。将rFPV-ILTV-gD和ILT弱毒疫苗同时免疫试验鸡,同时设非免疫对照鸡群。血清中和抗体检测结果表明:疫苗组与重组病毒组差异不显著(p＞0.05),而重组病毒组和疫苗组与空白对照组差异均显著(p＜0.05)。免疫42d攻毒试验表明,重组病毒组和疫苗组对ILTV强毒攻击的保护率均为96.67%,而空白对照组为6.67%。说明该重组病毒作为弱毒疫苗能够抵抗ILTV强毒的攻击,对免疫鸡群有较好的保护作用。
5.ILTV河南长葛株gD基因与鸡IL-2串联重组禽痘病毒的构建及免疫效力试验
将ILTV长葛株的gD基因与鸡IL-2串联到禽痘病毒表达载体中,转染后获得一株重组病毒rFPV-IL2-ILTV-gD,该重组病毒能够高效表达ILTV gD基因。将重组病毒rFPV-ILTV-gD、rFPV-IL2-ILTV-gD和ILT弱毒疫苗免疫试验动物;同时设空白对照。免疫后ELISA检测结果表明疫苗组与重组病毒rFPV-IL2-ILTV-gD组、重组病毒rFPV-IL2-ILTV-gD与重组病毒rFPV-ILTV-gD抗体效价相比差异不显著(p＞0.05),而重组病毒组rFPV-IL2-ILTV-gD、重组病毒rFPV-ILTV-gD和疫苗组与空白对照组抗体效价相比,差异均显著(p＜0.05)。免疫28d攻毒后每组鸡的发病情况表明重组病毒rFPV-IL2-ILTV-gD组、重组病毒rFPV-ILTV-gD和疫苗组对ILTV强毒攻击的保护率分别为96%、92%和96%,而空白对照组为8%。说明构建的重组病毒rFPV-IL2-ILTV-gD与商品弱毒疫苗的免疫效力相当,能够较好的保护免疫鸡群。
Infectious laryngotracheitis(ILT) is a highly contagious ILTV virus-induced an acute respiratory infection of chicken.Character of this disease is sign of respiratory depression, gasping,weight losses due to mortality and decreased egg production.The disease occurs worldwide and is one of the major health and economic problems in the poultry industry. ILT has been controlled efficiently by vaccination with modified live strains of ILTV.But all ILTV live vaccines remain certain virulence,and are able to establish latent infection for life time;the vaccine viruses may increase virulence during in vivo passage.Therefore, there is an urgent demand for safer vaccines which could be used to control and possibly eradicate ILTV.With development of the molecular biology,many researchers have made major commitment to rDNA-based vaccination.The fowl virus vaccine viruses are considered one of the most potent vector live vaccines expressing foreign antigens related to vaccine-induced immunity against poultry diseases.In this study,we constructed recombinant fowl virus with ChIL-2 and glycoprotein D of infectious laryngotracheitis virus of Hnchangge strain and evaluated the abilities of this recombinants to protect against mortality in SPF chickens.This study includes:
1.Isolation and Identification the Infectious Laryngotracheitis Virus HNchangge strain and Sequence Analysis of gB,gC and gD Gene
A virulent strain ILTV was isolated from the larynx and trachea tissue of infected chickens which was identification by double agar precipitation test.Full-length ILTV gB, gC and gD gene were amplified by PCR and then cloned into the pGEM-T vector.ILTV genes of gB,gC and gD were sequenced and the sequences were compared with the gB,gC and gD gene sequences from others strains of ILTV in GenBank.The nucleotide homology of ILTV gB,gC and gD gene were 99.7%～99.9%.It indicated that ILTV gB, gC and gD gene were conservative.
2.Construction of DNA Vaccines for ILTV HNchangge strain and their immune efficiency
Three genes encoding gB,gC and gD glycoprotein of ILTV HNchangge strain were inserted into pcDNA3.1 vector and named pcDNA-gB,pcDNA-gC and pcDNA-gD.The recombinant DNA vaccines were transcribed and expressed in eukaryotic cells effectively by IFA identification.Thirty30-day-old chickens were immunized by intramuscular injection with the DNA vaccines and blank vector pcDNA3.1,the dose of each injected plasmid for a chicken is 100μg.Another group chickens were vaccinated with the commercial ILT vaccine(D-805) and the blank control group chickens were injected with normal saline.The results of MTT assay showed that recombinant plasmid DNA induced strong proliferative response of Lymphocytes compared with the control groups.The serum-neutralization antibodies induced by DNA vaccine pcDNA-gB,pcDNA-gC and pcDNA-gD were higher than those induced by pcDNA3.1 vector.The results indicated that the recombinant plasmids pcDNA-gB,pcDNA-gC and pcDNA-gD had immune efficiency.
3.Expression of gD Gene of ILTV and Primary Development of Indirect ELISA Assay Based on the Expressed Protein
The gD gene of ILTV HNchangge strain was inserted into pET-28a(+) vector and named pET28-gD.The pET28-gD was further transformed into E.coli BL21 cells and a 55ku protein was high expressed after induced with IPTG.Western-blotting analysis proved the recombinant protein has a good reactive ability against ILT positive serum.The indirect ELISA for detecting ILTV antibodies was established by using purified recombinant gD protein.The optional working circumstances for the ELISA(antigen concentration of 24.5μg·mL,serum dilution of 1:320) were tried out with chess titration. The tested serum of positive criterion of this ELISA is OD_(450)≧0.25,the negative serum OD_(450)＜0.25.
4.Expression of glycoprotein D(gD) of ILTV in recombinant fowl pox virus and its immune efficacy
A recombinant fowl pox expressing glycoprotein gD named as rFPV-ILTV-gD was constructed by introducing gD gene of infectious laryngotracheitis virus isolated from Henan Changge recombined into the genome of fowl pox virus.Thirty-day-old non-immunized chickens were vaccinated with the rFPV-ILTV-gD and the commercial ILT vaccine(D-805).All groups were challenged with a lethal dose of virulent ILTV 42 days later.Results showed that protective efficacy could be significantly enhanced after immunizations and the anti-ILT neutralization antibody level were significantly increased compared with the unvaccinated chickens.There is no significant difference between the commercial vaccine and the rFPV-ILTV-gD considering the immune efficacy.Additionally, the recombined virus has the similar immune effects with the commercial ILT vaccine.
5.Construction of recombinant fowl pox virus with ChlL-2 and glyeoprotein D of ILTV and its immune efficacy
A recombinant fowl pox named as rFPV-IL2-ILTV-gD was constructed.Four groups of 20-day SPF chickens were immunized with sterilized water,commercial ILT vaccine (D-805),recombinant virus rFPV-ILTV-gD and recombinant virus rFPV-IL2-ILTV-gD respectively.Serum of chickens in each group was collected at 7,14,21,28,35,42,49,and 56 day post inoculation,and the antibody to ILT was measured by ELISA.Parts of chickens were challenged with a lethal dose of virulent ILTV at 28 day post inoculation.Results of immune protection test showed that there was no significant difference among the recombinant virus rFPV-ILTV-gD,the recombinant virus rFPV-IL2-ILTV-gD and the commercial vaccine considering the immune efficacy(P＞0.05),and the protection rates of the chickens after the virus challenge were 92%,96%and 96%,whereas that of the control group was 8%.It indicates,therefore,the recombined virus has similar immune potential to that of the commercial ILT vaccine,and can effectively protect chicken from ILTV challenge.
1.ILTV河南长葛株分离鉴定及其gB、gC和gD基因的克隆与序列分析
鸡胚尿囊膜接种和琼脂扩散试验对分离病毒进行鉴定,结果表明所分离病毒为ILTV,从而获得一株ILTV河南长葛株。并分别克隆其gB、gC和gD基因,测定其核苷酸序列,将其核苷酸与GenBank公布的ILTV相应序列进行分析和同源性比较,结果长葛分离株ILTV的gB、gC和gD基因3个序列分别与GenBank登录的ILTV gB、gC和gD基因序列存在很高的同源性,其同源性在99.7%～99.9%之间,说明ILTV的gB、gC和gD基因序列相对保守。
2.ILTV长葛株gB、gC和gD基因真核表达载体的构建及其免疫效果研究
将ILTV长葛株gB、gC和gD基因插入pcDNA3.1中,构建了真核表达载体pcDNA-gB、pcDNA-gC和pcDNA-gD。间接免疫荧光检测结果表明,三种重组质粒均能够在PK-15细胞中表达。以SPF鸡为试验动物,肌内注射构建的pcDNA-gB、pcDNA-gC、pcDNA-gD和pcDNA3.1空载体进行免疫接种。微量血清中和试验检测结果显示,构建的真核表达载体pcDNA-gB、pcDNA-gC、pcDNA-gD和活疫苗均能够诱导机体产生较高的抗体水平;MTT法检测表明构建的真核表达载体pcDNA-gB、pcDNA-gC、pcDNA-gD及活疫苗对照均能够诱导淋巴细胞增殖,同时也能够诱导强的CTL细胞的增殖。表明构建的载体pcDNA-gB、pcDNA-gC、pcDNA-gD和弱毒疫苗均能够有效诱导机体产生体液免疫和细胞免疫。
3.ILTV长葛株gD基因的表达及间接ELISA检测方法的初步建立
将ILTV gD基因插入到原核表达载体pET-28a(+)中,构建重组表达质粒pET28-gD。将pET28-gD转化到宿主表达菌BL21中,用IPTG诱导表达,SDS-PAGE结果显示表达目的蛋白大小为55ku,Western-blotting表明表达产物具有良好的免疫原性。表达的蛋白产物经纯化后作为ELISA包被抗原,初步建立了间接ELISA诊断方法,并确定了抗原最佳包被浓度为24.5μg·mL;最佳血清稀释度为1∶320,初步确定ELISA判定标准为OD_(450)≥0.25判为阳性,小于0.25为阴性。试验证明建立的间接ELISA具有很强的特异性和较高的敏感性且与多种病毒抗原无交叉反应。用初步建立的ELISA和微量血清中和试验检测临床送检的84份血清样品。结果两种方法的阳性符合率为96.5%(28/29),阴性符合率为98.2%(55/56),表明建立的方法可用于临床上ILT的检测。
4.表达ILTV长葛株gD基因重组禽痘病毒的构建及其免疫效力试验
将ILTV长葛株gD基因同源重组到禽痘病毒基因组中,得到能高效表达ILTV gD基因的重组禽痘病毒(rFPV-ILTV-gD)。将rFPV-ILTV-gD和ILT弱毒疫苗同时免疫试验鸡,同时设非免疫对照鸡群。血清中和抗体检测结果表明:疫苗组与重组病毒组差异不显著(p＞0.05),而重组病毒组和疫苗组与空白对照组差异均显著(p＜0.05)。免疫42d攻毒试验表明,重组病毒组和疫苗组对ILTV强毒攻击的保护率均为96.67%,而空白对照组为6.67%。说明该重组病毒作为弱毒疫苗能够抵抗ILTV强毒的攻击,对免疫鸡群有较好的保护作用。
5.ILTV河南长葛株gD基因与鸡IL-2串联重组禽痘病毒的构建及免疫效力试验
将ILTV长葛株的gD基因与鸡IL-2串联到禽痘病毒表达载体中,转染后获得一株重组病毒rFPV-IL2-ILTV-gD,该重组病毒能够高效表达ILTV gD基因。将重组病毒rFPV-ILTV-gD、rFPV-IL2-ILTV-gD和ILT弱毒疫苗免疫试验动物;同时设空白对照。免疫后ELISA检测结果表明疫苗组与重组病毒rFPV-IL2-ILTV-gD组、重组病毒rFPV-IL2-ILTV-gD与重组病毒rFPV-ILTV-gD抗体效价相比差异不显著(p＞0.05),而重组病毒组rFPV-IL2-ILTV-gD、重组病毒rFPV-ILTV-gD和疫苗组与空白对照组抗体效价相比,差异均显著(p＜0.05)。免疫28d攻毒后每组鸡的发病情况表明重组病毒rFPV-IL2-ILTV-gD组、重组病毒rFPV-ILTV-gD和疫苗组对ILTV强毒攻击的保护率分别为96%、92%和96%,而空白对照组为8%。说明构建的重组病毒rFPV-IL2-ILTV-gD与商品弱毒疫苗的免疫效力相当,能够较好的保护免疫鸡群。
Infectious laryngotracheitis(ILT) is a highly contagious ILTV virus-induced an acute respiratory infection of chicken.Character of this disease is sign of respiratory depression, gasping,weight losses due to mortality and decreased egg production.The disease occurs worldwide and is one of the major health and economic problems in the poultry industry. ILT has been controlled efficiently by vaccination with modified live strains of ILTV.But all ILTV live vaccines remain certain virulence,and are able to establish latent infection for life time;the vaccine viruses may increase virulence during in vivo passage.Therefore, there is an urgent demand for safer vaccines which could be used to control and possibly eradicate ILTV.With development of the molecular biology,many researchers have made major commitment to rDNA-based vaccination.The fowl virus vaccine viruses are considered one of the most potent vector live vaccines expressing foreign antigens related to vaccine-induced immunity against poultry diseases.In this study,we constructed recombinant fowl virus with ChIL-2 and glycoprotein D of infectious laryngotracheitis virus of Hnchangge strain and evaluated the abilities of this recombinants to protect against mortality in SPF chickens.This study includes:
1.Isolation and Identification the Infectious Laryngotracheitis Virus HNchangge strain and Sequence Analysis of gB,gC and gD Gene
A virulent strain ILTV was isolated from the larynx and trachea tissue of infected chickens which was identification by double agar precipitation test.Full-length ILTV gB, gC and gD gene were amplified by PCR and then cloned into the pGEM-T vector.ILTV genes of gB,gC and gD were sequenced and the sequences were compared with the gB,gC and gD gene sequences from others strains of ILTV in GenBank.The nucleotide homology of ILTV gB,gC and gD gene were 99.7%～99.9%.It indicated that ILTV gB, gC and gD gene were conservative.
2.Construction of DNA Vaccines for ILTV HNchangge strain and their immune efficiency
Three genes encoding gB,gC and gD glycoprotein of ILTV HNchangge strain were inserted into pcDNA3.1 vector and named pcDNA-gB,pcDNA-gC and pcDNA-gD.The recombinant DNA vaccines were transcribed and expressed in eukaryotic cells effectively by IFA identification.Thirty30-day-old chickens were immunized by intramuscular injection with the DNA vaccines and blank vector pcDNA3.1,the dose of each injected plasmid for a chicken is 100μg.Another group chickens were vaccinated with the commercial ILT vaccine(D-805) and the blank control group chickens were injected with normal saline.The results of MTT assay showed that recombinant plasmid DNA induced strong proliferative response of Lymphocytes compared with the control groups.The serum-neutralization antibodies induced by DNA vaccine pcDNA-gB,pcDNA-gC and pcDNA-gD were higher than those induced by pcDNA3.1 vector.The results indicated that the recombinant plasmids pcDNA-gB,pcDNA-gC and pcDNA-gD had immune efficiency.
3.Expression of gD Gene of ILTV and Primary Development of Indirect ELISA Assay Based on the Expressed Protein
The gD gene of ILTV HNchangge strain was inserted into pET-28a(+) vector and named pET28-gD.The pET28-gD was further transformed into E.coli BL21 cells and a 55ku protein was high expressed after induced with IPTG.Western-blotting analysis proved the recombinant protein has a good reactive ability against ILT positive serum.The indirect ELISA for detecting ILTV antibodies was established by using purified recombinant gD protein.The optional working circumstances for the ELISA(antigen concentration of 24.5μg·mL,serum dilution of 1:320) were tried out with chess titration. The tested serum of positive criterion of this ELISA is OD_(450)≧0.25,the negative serum OD_(450)＜0.25.
4.Expression of glycoprotein D(gD) of ILTV in recombinant fowl pox virus and its immune efficacy
A recombinant fowl pox expressing glycoprotein gD named as rFPV-ILTV-gD was constructed by introducing gD gene of infectious laryngotracheitis virus isolated from Henan Changge recombined into the genome of fowl pox virus.Thirty-day-old non-immunized chickens were vaccinated with the rFPV-ILTV-gD and the commercial ILT vaccine(D-805).All groups were challenged with a lethal dose of virulent ILTV 42 days later.Results showed that protective efficacy could be significantly enhanced after immunizations and the anti-ILT neutralization antibody level were significantly increased compared with the unvaccinated chickens.There is no significant difference between the commercial vaccine and the rFPV-ILTV-gD considering the immune efficacy.Additionally, the recombined virus has the similar immune effects with the commercial ILT vaccine.
5.Construction of recombinant fowl pox virus with ChlL-2 and glyeoprotein D of ILTV and its immune efficacy
A recombinant fowl pox named as rFPV-IL2-ILTV-gD was constructed.Four groups of 20-day SPF chickens were immunized with sterilized water,commercial ILT vaccine (D-805),recombinant virus rFPV-ILTV-gD and recombinant virus rFPV-IL2-ILTV-gD respectively.Serum of chickens in each group was collected at 7,14,21,28,35,42,49,and 56 day post inoculation,and the antibody to ILT was measured by ELISA.Parts of chickens were challenged with a lethal dose of virulent ILTV at 28 day post inoculation.Results of immune protection test showed that there was no significant difference among the recombinant virus rFPV-ILTV-gD,the recombinant virus rFPV-IL2-ILTV-gD and the commercial vaccine considering the immune efficacy(P＞0.05),and the protection rates of the chickens after the virus challenge were 92%,96%and 96%,whereas that of the control group was 8%.It indicates,therefore,the recombined virus has similar immune potential to that of the commercial ILT vaccine,and can effectively protect chicken from ILTV challenge.
引文
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