分别表达鸡白细胞介素1β、白细胞介素2和骨髓单核细胞生长因子基因重组鸡痘病毒的构建及其对ND疫苗免疫效力的影响
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摘要
在免疫反应发生部位，细胞因子微环境对抗原诱导免疫反应的质量和进程起重要作用。细胞因子介导的免疫调节作用可在免疫反应的不同阶段进行，如抗原提呈阶段；特异性免疫反应的效应阶段：免疫记忆反应建立阶段等。利用病毒载体在体内表达细胞因子具有如下优点：可延长细胞因子在体内存在的时间；依赖病毒的组织噬性，可使细胞因子到达特定部位：表达的重组细胞因子可保持更好的生物活性。本研究克隆鸡的白细胞介素1β(IL-1β)、白细胞介素2(IL-2)和骨髓单核细胞生长因子(MGF)的基因编码区；分别构建表达这3种细胞因子的重组鸡痘病毒(rFPVs)，通过检测它们在体外表达重组细胞因子的生物活性，证明rFPVs可以有效表达鸡IL-1β、IL-2和MGF；以SPF鸡和商品鸡为动物模型，研究rFPVs表达的鸡IL-1β、IL-2、IFN-γ和MGF对新城疫常规疫苗(LaSota)和基因工程疫苗(rFPV-HN)的免疫调节作用，探讨细胞因子发挥免疫佐剂作用的机理，以期开发一种新型的疫苗佐剂。
1 鸡IL-1β、IL-2和MGF基因的克隆及序列分析
在无菌条件下取出4周龄的SPF鸡脾脏，分离单核淋巴细胞，用含有伴刀豆蛋白(ConA)的培养液培养。分别在培养后的8h、24h、48h和72h收集脾淋巴细胞，提取细胞总RNA，分离纯化mRNA。根据GenBank上登录的鸡IL-1β(登录号：Y15006)、IL-2(登录号：AF00631)和MGF(登录号：X14477)cDNA序列，设计扩增鸡IL-1β、IL-2和MGF基因编码区的上、下游引物，在下游引物特异序列的5’端加上痘苗病毒转录终止信号的互补序列(AAA AAA AA)，用RT-PCR方法扩增鸡的IL-1β、IL-2和MGF基因编码区。PCR产物经凝胶电泳分析，扩增出的鸡IL-1β、IL-2和MGF基因编码区片段大小分别为804bp、432bp和606bp，并且发现ConA刺激的脾细胞持续表达IL-1β、IL-2和MGF的时间并
扬州大学博士学位论文
不相同，其中表达IL一lp持续的时间最长，表达MGF持续时间最短。测得的序列
与己发表的序列进行比较，发现鸡IL一1旦有4个核昔酸和1个氨基酸发生了变化，
IL一2有4个核昔酸和3个氨基酸发生了变化，而MGF的核昔酸和氨基酸没有发生
任何变化。这些位点的变化可能是同一种属不同品种间的正常变化，并不影响其
生物活性。
2分别表达鸡IL一lp、IL一2和MGF基因重组鸡痘病毒的构建及其表达
产物生物活性的检测
将扩增的鸡IL一1旦、IL一2和MGF基因编码区(带有痘苗病毒转录终止信号的
互补序列)克隆到鸡痘病毒(FPv)转移载体PI 175中，构建重组质粒pll751LI
p、Pll75ILZ和pll75MGF，使鸡IL一1日、IL一2和MGF基因编码区紧处于痘苗病
毒早晚期启动子p跳的下游。重组质粒pll75ILI日、pll75ILZ和pll75MGF分别
转染己感染FPV疫苗株282E4的鸡胚成纤维细胞中(CEF)，与FPV 282E4株发
生同源重组，通过蓝斑筛选和纯化，分别获得表达鸡IL一lp、IL一2和MGF基因
的重组鸡痘病毒(rFPVs)rFPV一IL lp、rFPV一ILZ和:FPV一MGF。将获得的rFPVs
分别感染单层CEF，72h收获细胞培养上清，经0.1林m滤膜除去其中的:FPvs;利
用XTT用MS法检测细胞上清中的重组细胞因子，效价分别为1 .0 xl了U/ml、3.6
x IO4uzml、2.1 x lo3U如l，证明吓Pvs可以很好地表达鸡IL一l日、IL一2和MGF。
3 rFPvs表达的鸡IL一lp、IL一2、IFN一Y和MGF对新城疫疫苗免疫效力
影响
用表达鸡IL一lp、IL一2、IFN一Y和MGF的:FPVs(rFPV一IL lp、rFPV一ILZ、rFPV-
IFNY和rFPV一MGF)接种不同日龄的SPF或商品鸡，同时免疫新城疫(ND)Lasota
疫苗或基因工程疫苗rFPV.HN，通过检测免疫后的体增重、抗体水平、脾脏中
CD矿T细胞和CDS+T细胞数量变化、攻毒后泄殖腔排毒率和保护率等相关免疫指
标，比较各组差异，评价细胞因子发挥免疫调节的效果，以期开发安全有效的新
型疫苗佐剂。
3.1 rFPVs表达的鸡细胞因子抵抗FPV抑制SPF雏鸡体重增长的作用
以SPF鸡为动物模型，FPV疫苗282E4(叭一FPV)可以明显抑制雏鸡早期体
重的增长，而:FPV表达的IL一2或IFN一Y可以显著抵抗FPV抑制体重增长:在一
定程度上，rFPV表达的IL一lp也表现出相似的作用，而接种表达MGF的:FPV试
邵卫星:rFPVs表达的鸡IL一lp、IL一2、IFN一丫或MGF对NO疫苗免疫效力影响川
验组鸡的体增重与野生鸡痘病毒(，比.FPV)对照组没有显著性差异。给鸡同时接
种表达几一2或IFN一丫的rFPVs，IL一2和IFN一Y不能协同增强抗FPV抑制体重增长。
可能是由于该组鸡接种的rFPV量最多，FPV的抑制体重增长的作用超过细胞因子
的免疫调节作用。
3.2 rFPVs表达的鸡细胞因子对ND疫苗诱导抗体生成的影响
rFPV表达的IL一lp、IL·2或MGF不能显著促进接种Lasota疫苗(1 pDso)的
SPF鸡产生抗NDV的HI抗体。用:FPV.HN免疫SPF鸡或NDV母源抗体低的商
品鸡，同时接种表达细胞因子的rFPvs，rFPv表达的IL一lp或IFN一丫在免疫后期
能促进抗HN间接ELISA抗体的滴度上升，而且产生抗体的整齐度好::FPV表达
的IL一2或MGF不能显著促进抗体的生成。联合应用IL一2和IFN一Y的实验组鸡虽
然产生抗HN间接ELISA抗体的滴度和整齐度比对照组好，但IL一2和IFN一Y对抗
体的产生并没有表现出协同增强作用。
33 rFPVs表达的鸡细胞因子对SPF鸡脾脏中CD4+T细胞?
Advances in understanding the cells and molecular interactions linking the innate immune system to adaptive immune response have revealed the critical role that cytokines serve in antigen presentation, cell activation and proliferation and the establishment of immune memory. Cytokines can enhance vaccine-induced immune responses at three levels: ?recruitment of immune reactive cells to inductive sites and activation of professional antigen presenting cells (APCs), (2) proliferation and differentiation of antigen specific T and B cells and enhancement of effector function and (3) transition of cells into the memory cell pool and lymphocyte homeostasis. As an alternative to the use of cytokine-inducing adjuvants, cytokines may also be used directly. Most cytokines have the ability to modify and re-direct immune responses. Virus vectors offer a very attractive means for the delivery of cytokines. Recombinant cytokines delivered by virus vectors can be expressed continuously and have a good biologic activity in
vivo. In the present study, the genes of chicken interleukin 1 B (IL-1 B), interleukin 2 (IL-2) and myelomonocytic growth factor (MGF) were amplified by RT-PCR from spleen cells stimulated by ConA. We constructed recombinant fowlpox viruses (rFPVs) expressing chicken IL-1 3 , IL-2 and MGF. Four chicken cytokines (IL-1 P , IL-2, IFN- Y , MGF) expressed by rFPVs were evaluated for their adjuvant effects on immune responses in SPF chickens and commercial chickens vaccinated with suboptimal dose of La Sota virus or minimal immune dose of rFPV expressing Newcastle disease virus (NDV) HN gene (rFPV-HN). We hope to explore a novel vaccine adjuvant.
1. Cloning of genes of chicken IL-1 0 , IL-2 and MGF
Spleen cells from 4-wk-old chicken were cultured with ConA (10ulml) in RPMI 1640 containing 5% FCS at a concentration of 107 cells/ml. Cells were cultured for 8h, 24h, 48h or 72h respectively at 41 C in 5% CO2 incubator. Total RNAs were extracted from cells cultured for different times and mRNA were isolated from total RNA. The sequences of the PCR primers were designed on the basis of published cDNA sequences of chicken IL-1 B , IL-2 or MGF. The 5' end of downstream primers contained a sequence (AAAAAAAAA) which is the complement sequence of transcriptional terminal signal of vaccinia virus. The PCR reaction mixture was analyzed by agrose gel electrophoresis after amplification. The results showed that the purposed genes were obtained. At the same time we found that chicken IL-1 B , IL-2 or MGF was expressed by ConA-stimulated spleen cells at various time. The time of expression of IL-1 B was the longest and MGF was the shortest. The cloned sequences of chicken IL-1 3 had a change of four nucleotide sites and one amino acid site and the chicken IL-2 had a variation of four and three sites respectively compared with the published sequences. The change of these sites would be the normal variation in different species and have no effect on their biologic activity.
2. Construction of rFPVs expressing chicken IL-1B , IL-2 and MGF and assay of biologic activity of the product in vitro
The sequences encoding chicken IL-1 B , IL-2 and MGF were inserted into FPV (282E4) using the transfer plasmid p1175 under the control of the FPV early/late promoter (PE/L)- The plasmid p1175 contains the E.coli LacZ gene under the control of the PFV late promoter (P11), enabling rapid identification of FPV recombinants. Primary CEF monolayers were infected with rFPVs expressing chicken IL-1 3 , IL-2 and MGF at a M.O.I of 1.0. Following incubation at 37C for 72h, culture supernatants were harvested and filtered through a 0.1 um filter to remove infectious FPV. The levels of chicken IL-1 3 , IL-2 or MGF in cell culture supernatants infected with rFPVs were determined according to the standard procedures of XTT/PMS method. The results showed that rFPVs expressed chicken IL-1 3 , IL-2 and MGF effectively.
3. Influence of chicken IL-1 B, IL-2, IFN- Y and MGF expressed by rFPVs on vaccination of La Sota or rFPV-HN against Newcastle disease vir
1 鸡IL-1β、IL-2和MGF基因的克隆及序列分析
在无菌条件下取出4周龄的SPF鸡脾脏，分离单核淋巴细胞，用含有伴刀豆蛋白(ConA)的培养液培养。分别在培养后的8h、24h、48h和72h收集脾淋巴细胞，提取细胞总RNA，分离纯化mRNA。根据GenBank上登录的鸡IL-1β(登录号：Y15006)、IL-2(登录号：AF00631)和MGF(登录号：X14477)cDNA序列，设计扩增鸡IL-1β、IL-2和MGF基因编码区的上、下游引物，在下游引物特异序列的5’端加上痘苗病毒转录终止信号的互补序列(AAA AAA AA)，用RT-PCR方法扩增鸡的IL-1β、IL-2和MGF基因编码区。PCR产物经凝胶电泳分析，扩增出的鸡IL-1β、IL-2和MGF基因编码区片段大小分别为804bp、432bp和606bp，并且发现ConA刺激的脾细胞持续表达IL-1β、IL-2和MGF的时间并
扬州大学博士学位论文
不相同，其中表达IL一lp持续的时间最长，表达MGF持续时间最短。测得的序列
与己发表的序列进行比较，发现鸡IL一1旦有4个核昔酸和1个氨基酸发生了变化，
IL一2有4个核昔酸和3个氨基酸发生了变化，而MGF的核昔酸和氨基酸没有发生
任何变化。这些位点的变化可能是同一种属不同品种间的正常变化，并不影响其
生物活性。
2分别表达鸡IL一lp、IL一2和MGF基因重组鸡痘病毒的构建及其表达
产物生物活性的检测
将扩增的鸡IL一1旦、IL一2和MGF基因编码区(带有痘苗病毒转录终止信号的
互补序列)克隆到鸡痘病毒(FPv)转移载体PI 175中，构建重组质粒pll751LI
p、Pll75ILZ和pll75MGF，使鸡IL一1日、IL一2和MGF基因编码区紧处于痘苗病
毒早晚期启动子p跳的下游。重组质粒pll75ILI日、pll75ILZ和pll75MGF分别
转染己感染FPV疫苗株282E4的鸡胚成纤维细胞中(CEF)，与FPV 282E4株发
生同源重组，通过蓝斑筛选和纯化，分别获得表达鸡IL一lp、IL一2和MGF基因
的重组鸡痘病毒(rFPVs)rFPV一IL lp、rFPV一ILZ和:FPV一MGF。将获得的rFPVs
分别感染单层CEF，72h收获细胞培养上清，经0.1林m滤膜除去其中的:FPvs;利
用XTT用MS法检测细胞上清中的重组细胞因子，效价分别为1 .0 xl了U/ml、3.6
x IO4uzml、2.1 x lo3U如l，证明吓Pvs可以很好地表达鸡IL一l日、IL一2和MGF。
3 rFPvs表达的鸡IL一lp、IL一2、IFN一Y和MGF对新城疫疫苗免疫效力
影响
用表达鸡IL一lp、IL一2、IFN一Y和MGF的:FPVs(rFPV一IL lp、rFPV一ILZ、rFPV-
IFNY和rFPV一MGF)接种不同日龄的SPF或商品鸡，同时免疫新城疫(ND)Lasota
疫苗或基因工程疫苗rFPV.HN，通过检测免疫后的体增重、抗体水平、脾脏中
CD矿T细胞和CDS+T细胞数量变化、攻毒后泄殖腔排毒率和保护率等相关免疫指
标，比较各组差异，评价细胞因子发挥免疫调节的效果，以期开发安全有效的新
型疫苗佐剂。
3.1 rFPVs表达的鸡细胞因子抵抗FPV抑制SPF雏鸡体重增长的作用
以SPF鸡为动物模型，FPV疫苗282E4(叭一FPV)可以明显抑制雏鸡早期体
重的增长，而:FPV表达的IL一2或IFN一Y可以显著抵抗FPV抑制体重增长:在一
定程度上，rFPV表达的IL一lp也表现出相似的作用，而接种表达MGF的:FPV试
邵卫星:rFPVs表达的鸡IL一lp、IL一2、IFN一丫或MGF对NO疫苗免疫效力影响川
验组鸡的体增重与野生鸡痘病毒(，比.FPV)对照组没有显著性差异。给鸡同时接
种表达几一2或IFN一丫的rFPVs，IL一2和IFN一Y不能协同增强抗FPV抑制体重增长。
可能是由于该组鸡接种的rFPV量最多，FPV的抑制体重增长的作用超过细胞因子
的免疫调节作用。
3.2 rFPVs表达的鸡细胞因子对ND疫苗诱导抗体生成的影响
rFPV表达的IL一lp、IL·2或MGF不能显著促进接种Lasota疫苗(1 pDso)的
SPF鸡产生抗NDV的HI抗体。用:FPV.HN免疫SPF鸡或NDV母源抗体低的商
品鸡，同时接种表达细胞因子的rFPvs，rFPv表达的IL一lp或IFN一丫在免疫后期
能促进抗HN间接ELISA抗体的滴度上升，而且产生抗体的整齐度好::FPV表达
的IL一2或MGF不能显著促进抗体的生成。联合应用IL一2和IFN一Y的实验组鸡虽
然产生抗HN间接ELISA抗体的滴度和整齐度比对照组好，但IL一2和IFN一Y对抗
体的产生并没有表现出协同增强作用。
33 rFPVs表达的鸡细胞因子对SPF鸡脾脏中CD4+T细胞?
Advances in understanding the cells and molecular interactions linking the innate immune system to adaptive immune response have revealed the critical role that cytokines serve in antigen presentation, cell activation and proliferation and the establishment of immune memory. Cytokines can enhance vaccine-induced immune responses at three levels: ?recruitment of immune reactive cells to inductive sites and activation of professional antigen presenting cells (APCs), (2) proliferation and differentiation of antigen specific T and B cells and enhancement of effector function and (3) transition of cells into the memory cell pool and lymphocyte homeostasis. As an alternative to the use of cytokine-inducing adjuvants, cytokines may also be used directly. Most cytokines have the ability to modify and re-direct immune responses. Virus vectors offer a very attractive means for the delivery of cytokines. Recombinant cytokines delivered by virus vectors can be expressed continuously and have a good biologic activity in
vivo. In the present study, the genes of chicken interleukin 1 B (IL-1 B), interleukin 2 (IL-2) and myelomonocytic growth factor (MGF) were amplified by RT-PCR from spleen cells stimulated by ConA. We constructed recombinant fowlpox viruses (rFPVs) expressing chicken IL-1 3 , IL-2 and MGF. Four chicken cytokines (IL-1 P , IL-2, IFN- Y , MGF) expressed by rFPVs were evaluated for their adjuvant effects on immune responses in SPF chickens and commercial chickens vaccinated with suboptimal dose of La Sota virus or minimal immune dose of rFPV expressing Newcastle disease virus (NDV) HN gene (rFPV-HN). We hope to explore a novel vaccine adjuvant.
1. Cloning of genes of chicken IL-1 0 , IL-2 and MGF
Spleen cells from 4-wk-old chicken were cultured with ConA (10ulml) in RPMI 1640 containing 5% FCS at a concentration of 107 cells/ml. Cells were cultured for 8h, 24h, 48h or 72h respectively at 41 C in 5% CO2 incubator. Total RNAs were extracted from cells cultured for different times and mRNA were isolated from total RNA. The sequences of the PCR primers were designed on the basis of published cDNA sequences of chicken IL-1 B , IL-2 or MGF. The 5' end of downstream primers contained a sequence (AAAAAAAAA) which is the complement sequence of transcriptional terminal signal of vaccinia virus. The PCR reaction mixture was analyzed by agrose gel electrophoresis after amplification. The results showed that the purposed genes were obtained. At the same time we found that chicken IL-1 B , IL-2 or MGF was expressed by ConA-stimulated spleen cells at various time. The time of expression of IL-1 B was the longest and MGF was the shortest. The cloned sequences of chicken IL-1 3 had a change of four nucleotide sites and one amino acid site and the chicken IL-2 had a variation of four and three sites respectively compared with the published sequences. The change of these sites would be the normal variation in different species and have no effect on their biologic activity.
2. Construction of rFPVs expressing chicken IL-1B , IL-2 and MGF and assay of biologic activity of the product in vitro
The sequences encoding chicken IL-1 B , IL-2 and MGF were inserted into FPV (282E4) using the transfer plasmid p1175 under the control of the FPV early/late promoter (PE/L)- The plasmid p1175 contains the E.coli LacZ gene under the control of the PFV late promoter (P11), enabling rapid identification of FPV recombinants. Primary CEF monolayers were infected with rFPVs expressing chicken IL-1 3 , IL-2 and MGF at a M.O.I of 1.0. Following incubation at 37C for 72h, culture supernatants were harvested and filtered through a 0.1 um filter to remove infectious FPV. The levels of chicken IL-1 3 , IL-2 or MGF in cell culture supernatants infected with rFPVs were determined according to the standard procedures of XTT/PMS method. The results showed that rFPVs expressed chicken IL-1 3 , IL-2 and MGF effectively.
3. Influence of chicken IL-1 B, IL-2, IFN- Y and MGF expressed by rFPVs on vaccination of La Sota or rFPV-HN against Newcastle disease vir
引文
1．巴德年主编．当代免疫学技术与应用．北京：北京医科大学中国协和医科大学联合出版社，1998．49-71．
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