堆型艾美耳球虫乳酸脱氢酶DNA疫苗免疫保护作用及免疫机理研究
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摘要
鸡球虫病是由艾美耳属(Eimeria)球虫寄生于鸡肠上皮细胞内所引起的寄生虫病,分布于世界各地,对养禽业危害巨大。由于目前的药物预防、活疫苗接种等防控措施存在较多问题,因此,有必要研究新的防控手段。DNA疫苗可以诱导宿主产生长期的体液免疫和细胞免疫,预防包括球虫病在内的多种疾病,因此成为疫苗发展的新方向。已经有多种球虫保护性抗原如EtMICs、SO7、5401、cSZ-1、3-1E等被发现,其保护效果也已经被大量的研究所证实。研究发现,堆型艾美耳球虫(E. acervulina)乳酸脱氢酶(LDH)是相对分子质量为37 kDa的蛋白,在球虫发育的4个阶段(卵囊、子孢子、裂殖体和裂殖子)均有相似水平的表达,可对E. acervulina的攻击产生部分保护力,被认为是一种很有潜力的疫苗候选抗原。
本研究利用RT-PCR方法从E. acervulina第一代裂殖体克隆了E. acervulina LDH基因,酶切及PCR鉴定均正确的菌株经测序,结果显示,克隆片段含一个长993 bp的开放阅读框(ORF),编码330个氨基酸,与GenBank上的E. acervulina LDH基因序列相似性为98%。应用DNA重组技术,将LDH基因克隆到原核表达载体pET28a(+)中,转化大肠杆菌BL21(DE3),通过IPTG诱导表达重组LDH蛋白。SDS-PAGE电泳显示,pET28a(+)-LDH在大肠杆菌中得到成功表达,其分子量在41 kDa左右。菌体经超声破碎后的沉淀与上清的SDS-PAGE分析结果表明,重组蛋白主要以包涵体形式存在。
本研究利用DNA重组技术,分别构建pVAX-LDH、pVAX-LDH-IFN-γ和pVAX-LDH-IL-2 DNA疫苗。经鉴定正确后,分别用重组质粒免疫14日龄雏鸡,1周后取肌肉注射部位、非注射部位组织和pVAX1空质粒注射部位,用RT-PCR和western blot检测疫苗的转录和表达情况。结果表明,构建的DNA疫苗均能在注射部位肌肉成功转录和表达。
将构建的DNA疫苗pVAX-LDH、pVAX-LDH-IFN-γ和pVAX-LDH-IL-2以及LDH重组蛋白和包涵体经腿部肌肉分别于14、21日龄两次免疫雏鸡,并设立感染非免疫和非感染非免疫对照组,另设pVAX1空质粒注射组。除非感染非免疫组外,28日龄经口接种新鲜的E. acervulina孢子化卵囊,攻虫第6天宰杀全部实验动物,计算存活率、进行病变记分、增重、卵囊下降率,通过计算综合抗球虫指数(ACI)这些生理指标,评价构建的疫苗对鸡抗球虫保护力。结果表明,所构建的重组质粒对感染球虫鸡均具有一定的保护效果,其中以pVAX-LDH-IFN-γ和pVAX-LDH-IL-2重组质粒组保护效果最好。
用构建的3种DNA疫苗免疫14日龄雏鸡,并设灭菌水注射和pVAX1空质粒对照组。21日龄以同样剂量加强免疫一次,二免后第7天,取鸡脾脏分离淋巴细胞。采用MTT法测定脾脏淋巴细胞增殖反应。结果发现,DNA疫苗免疫后鸡脾脏淋巴细胞增殖水平均显著高于灭菌水和pVAX1空质粒对照组。说明构建的DNA疫苗可以刺激鸡体的免疫系统尤其是细胞免疫反应。
用构建的3种DNA疫苗免疫14日龄雏鸡,并设灭菌水注射和pVAX1空质粒对照组,21日龄以同样剂量加强免疫一次。分别于首次免疫后第7天和第2次免疫后第7天取鸡脾脏和盲肠扁桃体分离淋巴细胞,用带有荧光标记的CD3+、CD4+、CD8+单克隆抗体进行荧光染色,用流式细胞术检测CD3+、CD4+/CD3+、CD8+/CD3+的比例。结果发现,第1次免疫后第7天脾脏和盲肠扁桃体中各组间CD3+ T淋巴细胞水平差异均不显著(P>0.05),第2次免疫后第7天,DNA疫苗组CD3+水平显著高于灭菌水和pVAX1空质粒组(P<0.05)。结果表明,构建的DNA疫苗可以增强鸡体T淋巴细胞反应。
用构建的3种DNA疫苗免疫14日龄雏鸡,并设灭菌水注射和pVAX1空质粒对照组,21日龄以同样剂量加强免疫一次。二免后第7天,取鸡脾脏和盲肠扁桃体分离淋巴细胞。提取淋巴细胞总RNA,反转录合成cDNA,进行实时荧光定量PCR,检测脾脏和盲肠扁桃体中IFN-γ、IL-2、IL-4、TNFSF15、IL-17D和TGF-β4 mRNA水平变化,以GAPDH为内参照,采用2-ΔΔCT方法计算各细胞因子相对mRNA表达量。结果发现,疫苗免疫后鸡脾脏和盲肠扁桃体IFN-γ、IL-2、TNFSF15、IL-17D和TGF-β4 mRNA水平显著升高,表明.,本研究构建的DNA疫苗可以促进鸡体内细胞因子分泌。
用构建的3种DNA疫苗免疫14日龄雏鸡,并设灭菌水注射和pVAX1空质粒对照组,21日龄以同样剂量加强免疫一次。1周后心脏采全血,制备血清,之后每间隔1周心脏采血,一直采样到二免后第6周。用重组LDH蛋白作为包被抗原,采用间接ELISA方法,检测血清样品的IgG水平。结果发现：二免后1～6周,疫苗组显示了显著高于灭菌水和pVAX1空质粒组的特异性LDH抗体水平。pVAX-LDH-IFN-γ组和pVAX-LDH-IL-2组的抗体滴度比pVAX-LDH高,且差异显著。pVAX-LDH-IL-2组在3个疫苗组中表现最高的抗体滴度。在整个过程中,注射灭菌水和pVAX1空质粒的两个组均未检测到特异性的抗体。结果表明,构建的DNA疫苗可以诱导鸡体产生特异性抗体。
本研究通过动物保护性试验和免疫机理研究结果证实,构建的DNA疫苗可以刺激机体产生显著的全身和局部免疫反应,诱导机体产生细胞免疫和体液免疫共同参与抵抗球虫感染,为机体提供一定保护力。
Avian coccidiosis, caused by intestinal infection with Eimeria spp. invading intraepithelial cells, is spread worldwide and economically the most important parasitic disease of the poultry industry. At present, the controls of Eimeria infections are still mainly based on the anticoccidial drugs and live vaccines. However, these measures have many drawbacks. It is necessary to develop alternative control strategies against coccidiosis. DNA vaccine has been confirmed that it can provoke both humoral and cell-mediated immune responses and prevent various diseases including coccidiosis. It shows a new way to develop vaccine. A few of coccidial protective antigens such as EtMICs, SO7,5401, cSZ-1, 3-1E have been identified and their protective effects have been confirmed. A 37 kDa protein of E. acervulina lactate dehydrogenase of was identified that was partially protective and considered an vaccine antigen candidate. This protein was present at similar levels throughout four different parasitic stages (oocysts, sporozoites, schizonts and merozoites).
In this study, E. acervulina LDH gene was amplified by RT-PCR from the first generation schizonts of E. acervulina. The ORF of E. acervulina LDH had 993 nucleotides and was predicted to encode a protein of 37 kDa. After confirm by PCR amplification, endonuclease cleavage and sequence analysis, the results showed that fragment had the open reading frame (ORF) of 993 bp in length and encoded 330 amino acids, which shared nucleotide sequence similarity of 98% with that of E. acervulina LDH in GenBank. By the means of DNA recombination technique, LDH gene was cloned into prokaryotic expression vector pET28a (+) and transformed into BL21 (DE3). The recombinant protein was expressed by inducing with IPTG As the results of SDS-PAGE indicated, the recombinant protein was successfully expressed in E. coli with the relative molecular weight of about 41 kDa. The results of SDS-PAGE of the pellet and supernatant after ultrasonication showed that most of the recombinant protein existed in the inclusion body.
By the means of DNA recombination technique, three DNA vaccines pVAX-LDH, pVAX-LDH-IFN-γand pVAX-LDH-IL-2 were constructed. Two-week-old chickens were injected intramuscularly (IM) in leg muscle with recombinant plasmids after they were identified. One week post-inoculation, vaccine injected tissues, non-injected tissues and pVAX1 plasmid injected tissues were collected. Expressions of proteins encoded by plasmids DNA in vivo were detected by reverse transcription-polymerase chain reaction (RT-PCR) and western blot assay. Results indicated that all DNA vaccines could be well transcripted and expressed in injected tissues.
Two-week-old chickens were inoculated with DNA vaccines pVAX-LDH, pVAX-LDH-IFN-γ, pVAX-LDH-IL-2, recombinant LDH protein and inclusion bodies by leg intramuscular injection, respectively. A booster immunization was given 1 week later. Challenged control group and unchallenged control chickens were injected with sterile TE buffer at the same injection site. pVAX1 plasmid alone was given to chickens as plasmid control. All chickens except the unchallenged control group were challenged orally with 1.2×105 sporulated oocysts of E. acervulina 7 days following second injection. All chickens were euthanized to determine the effects of immunization on the 6th day post-challenge. The efficacy of immunization was evaluated on the basis of survival rate, lesion score, body weight gain, oocyst decrease ratio and anti-coccidial index (ACI). The results indicated the DNA vaccines could protect chickens from E. acervulina infection and pVAX-LDH-IFN-γand pVAX-LDH-IL-2 could induce better protection.
Two-week-old chickens were inoculated with DNA vaccines pVAX-LDH, pVAX-LDH-IFN-γ, pVAX-LDH-IL-2, respectively. A booster immunization was given 1 week later. Plasmid control group chickens and water control group were given pVAXl plasmid alone and sterile water, respectively. On day 7 post-second immunization, spleens were removed from five chickens and lymphocytes were prepared. Lymphocyte proliferation responses were determined by MTT. The results showed that splenocytes from DNA vaccinated chickens displayed significantly greater proliferation compared with plasmid control group chickens and water control group. This indicated that DNA vaccines constructed in this study could stimulate the immune system especially cell-mediated response of chickens.
Two-week-old chickens were inoculated with DNA vaccines pVAX-LDH, pVAX-LDH-IFN-γ, pVAX-LDH-IL-2, respectively. A booster immunization was given 1 week later. Plasmid control group chickens and water control group were given pVAX1 plasmid alone and sterile water, respectively. On day 7 and 14 post primary immunization, spleens and cecal tonsils were removed from five chickens of each group. Splenocytes were dually stained with mouse anti-chicken CD3+, CD4+ and CD8+ monoclonal antibody. Percentage of CD3+,CD4+/CD3+ and CD8+/CD3+ were determined by flow cytometric analysis. The results showed that percentage of CD3+T lymphocytes in lymphocytes obtained from spleen and cecal tonsil 7 days post primary injection showed no differences (p>0.05). However, on day 7 following booster, percentages of CD3+T cells of vaccinated groups in both spleen and cecal tonsil were significantly higher than that of control groups (p<0.05). This indicated that DNA vaccines constructed in this study could enhance T lymphocytes response of chickens.
Two-week-old chickens were inoculated with DNA vaccines pVAX-LDH, pVAX-LDH-IFN-γ, pVAX-LDH-IL-2, respectively. A booster immunization was given 1 week later. Plasmid control group chickens and water control group were given pVAX1 plasmid alone and sterile water, respectively. On day 7 post-second immunization, spleens and cecal tonsils were removed from chickens and lymphocytes were prepared. Total RNA was extracted from lymphocytes and cDNA was synthesized. The changes of IFN-γ, IL-2, IL-4, TNFSF15, IL-17D, TGF-β4 mRNA were determined by real-time PCR. The relative quantification of cytokine gene mRNA compared with the internal control gene GAPDH was calculated using 2-ΔΔCT method. The results showed that the levels of IFN-γ, IL-2, TNFSF15, IL-17D and TGF-β4 mRNA in spleens and cecal tonsils of chickens were significantly higher following DNA vaccination. This indicated that DNA vaccines constructed in this study could promote the secretion of cytokines.
Two-week-old chickens were inoculated with DNA vaccines pVAX-LDH, pVAX-LDH-IFN-γ, pVAX-LDH-IL-2, respectively. A booster immunization was given 1 week later. Plasmid control group chickens and water control group were given pVAX1 plasmid alone and sterile water, respectively. Whole blood from chicks was collected by cardiac puncture at weeks 1,2,3,4,5 and 6 post-second immunization and the serum was collected. The serum IgG antibody levels were determined by ELISA and E. acervulina LDH recombinant protein was used as coating antigen. The results showed that anti-LDH antibody titers were higher in chickens vaccinated with DNA vaccines compared to those of water control and plasmid control chickens during weeks 1-6 post-second immunization (P <0.05). Antibody titers were significantly greater in groups vaccinated with pVAX-LDH-IFN-γand pVAX-LDH-IL-2 than group immunized with pVAX-LDH (P<0.05). Chickens vaccinated with pVAX-LDH-IL-2 developed the highest antibody titer compared with the other two vaccinated groups. Non-specific antibody was detected in water control and plasmid control chickens throughout the experiment. These results indicated that DNA vaccines could induce specific antibody in chickens.
The results of immune protective effects and mechanism of DNA vaccines on chickens showed that DNA vaccines could simulate the systemic and local immune system of chickens and induce both cell-mediated and humoral responses to resist coccidiosis.
本研究利用RT-PCR方法从E. acervulina第一代裂殖体克隆了E. acervulina LDH基因,酶切及PCR鉴定均正确的菌株经测序,结果显示,克隆片段含一个长993 bp的开放阅读框(ORF),编码330个氨基酸,与GenBank上的E. acervulina LDH基因序列相似性为98%。应用DNA重组技术,将LDH基因克隆到原核表达载体pET28a(+)中,转化大肠杆菌BL21(DE3),通过IPTG诱导表达重组LDH蛋白。SDS-PAGE电泳显示,pET28a(+)-LDH在大肠杆菌中得到成功表达,其分子量在41 kDa左右。菌体经超声破碎后的沉淀与上清的SDS-PAGE分析结果表明,重组蛋白主要以包涵体形式存在。
本研究利用DNA重组技术,分别构建pVAX-LDH、pVAX-LDH-IFN-γ和pVAX-LDH-IL-2 DNA疫苗。经鉴定正确后,分别用重组质粒免疫14日龄雏鸡,1周后取肌肉注射部位、非注射部位组织和pVAX1空质粒注射部位,用RT-PCR和western blot检测疫苗的转录和表达情况。结果表明,构建的DNA疫苗均能在注射部位肌肉成功转录和表达。
将构建的DNA疫苗pVAX-LDH、pVAX-LDH-IFN-γ和pVAX-LDH-IL-2以及LDH重组蛋白和包涵体经腿部肌肉分别于14、21日龄两次免疫雏鸡,并设立感染非免疫和非感染非免疫对照组,另设pVAX1空质粒注射组。除非感染非免疫组外,28日龄经口接种新鲜的E. acervulina孢子化卵囊,攻虫第6天宰杀全部实验动物,计算存活率、进行病变记分、增重、卵囊下降率,通过计算综合抗球虫指数(ACI)这些生理指标,评价构建的疫苗对鸡抗球虫保护力。结果表明,所构建的重组质粒对感染球虫鸡均具有一定的保护效果,其中以pVAX-LDH-IFN-γ和pVAX-LDH-IL-2重组质粒组保护效果最好。
用构建的3种DNA疫苗免疫14日龄雏鸡,并设灭菌水注射和pVAX1空质粒对照组。21日龄以同样剂量加强免疫一次,二免后第7天,取鸡脾脏分离淋巴细胞。采用MTT法测定脾脏淋巴细胞增殖反应。结果发现,DNA疫苗免疫后鸡脾脏淋巴细胞增殖水平均显著高于灭菌水和pVAX1空质粒对照组。说明构建的DNA疫苗可以刺激鸡体的免疫系统尤其是细胞免疫反应。
用构建的3种DNA疫苗免疫14日龄雏鸡,并设灭菌水注射和pVAX1空质粒对照组,21日龄以同样剂量加强免疫一次。分别于首次免疫后第7天和第2次免疫后第7天取鸡脾脏和盲肠扁桃体分离淋巴细胞,用带有荧光标记的CD3+、CD4+、CD8+单克隆抗体进行荧光染色,用流式细胞术检测CD3+、CD4+/CD3+、CD8+/CD3+的比例。结果发现,第1次免疫后第7天脾脏和盲肠扁桃体中各组间CD3+ T淋巴细胞水平差异均不显著(P>0.05),第2次免疫后第7天,DNA疫苗组CD3+水平显著高于灭菌水和pVAX1空质粒组(P<0.05)。结果表明,构建的DNA疫苗可以增强鸡体T淋巴细胞反应。
用构建的3种DNA疫苗免疫14日龄雏鸡,并设灭菌水注射和pVAX1空质粒对照组,21日龄以同样剂量加强免疫一次。二免后第7天,取鸡脾脏和盲肠扁桃体分离淋巴细胞。提取淋巴细胞总RNA,反转录合成cDNA,进行实时荧光定量PCR,检测脾脏和盲肠扁桃体中IFN-γ、IL-2、IL-4、TNFSF15、IL-17D和TGF-β4 mRNA水平变化,以GAPDH为内参照,采用2-ΔΔCT方法计算各细胞因子相对mRNA表达量。结果发现,疫苗免疫后鸡脾脏和盲肠扁桃体IFN-γ、IL-2、TNFSF15、IL-17D和TGF-β4 mRNA水平显著升高,表明.,本研究构建的DNA疫苗可以促进鸡体内细胞因子分泌。
用构建的3种DNA疫苗免疫14日龄雏鸡,并设灭菌水注射和pVAX1空质粒对照组,21日龄以同样剂量加强免疫一次。1周后心脏采全血,制备血清,之后每间隔1周心脏采血,一直采样到二免后第6周。用重组LDH蛋白作为包被抗原,采用间接ELISA方法,检测血清样品的IgG水平。结果发现：二免后1～6周,疫苗组显示了显著高于灭菌水和pVAX1空质粒组的特异性LDH抗体水平。pVAX-LDH-IFN-γ组和pVAX-LDH-IL-2组的抗体滴度比pVAX-LDH高,且差异显著。pVAX-LDH-IL-2组在3个疫苗组中表现最高的抗体滴度。在整个过程中,注射灭菌水和pVAX1空质粒的两个组均未检测到特异性的抗体。结果表明,构建的DNA疫苗可以诱导鸡体产生特异性抗体。
本研究通过动物保护性试验和免疫机理研究结果证实,构建的DNA疫苗可以刺激机体产生显著的全身和局部免疫反应,诱导机体产生细胞免疫和体液免疫共同参与抵抗球虫感染,为机体提供一定保护力。
Avian coccidiosis, caused by intestinal infection with Eimeria spp. invading intraepithelial cells, is spread worldwide and economically the most important parasitic disease of the poultry industry. At present, the controls of Eimeria infections are still mainly based on the anticoccidial drugs and live vaccines. However, these measures have many drawbacks. It is necessary to develop alternative control strategies against coccidiosis. DNA vaccine has been confirmed that it can provoke both humoral and cell-mediated immune responses and prevent various diseases including coccidiosis. It shows a new way to develop vaccine. A few of coccidial protective antigens such as EtMICs, SO7,5401, cSZ-1, 3-1E have been identified and their protective effects have been confirmed. A 37 kDa protein of E. acervulina lactate dehydrogenase of was identified that was partially protective and considered an vaccine antigen candidate. This protein was present at similar levels throughout four different parasitic stages (oocysts, sporozoites, schizonts and merozoites).
In this study, E. acervulina LDH gene was amplified by RT-PCR from the first generation schizonts of E. acervulina. The ORF of E. acervulina LDH had 993 nucleotides and was predicted to encode a protein of 37 kDa. After confirm by PCR amplification, endonuclease cleavage and sequence analysis, the results showed that fragment had the open reading frame (ORF) of 993 bp in length and encoded 330 amino acids, which shared nucleotide sequence similarity of 98% with that of E. acervulina LDH in GenBank. By the means of DNA recombination technique, LDH gene was cloned into prokaryotic expression vector pET28a (+) and transformed into BL21 (DE3). The recombinant protein was expressed by inducing with IPTG As the results of SDS-PAGE indicated, the recombinant protein was successfully expressed in E. coli with the relative molecular weight of about 41 kDa. The results of SDS-PAGE of the pellet and supernatant after ultrasonication showed that most of the recombinant protein existed in the inclusion body.
By the means of DNA recombination technique, three DNA vaccines pVAX-LDH, pVAX-LDH-IFN-γand pVAX-LDH-IL-2 were constructed. Two-week-old chickens were injected intramuscularly (IM) in leg muscle with recombinant plasmids after they were identified. One week post-inoculation, vaccine injected tissues, non-injected tissues and pVAX1 plasmid injected tissues were collected. Expressions of proteins encoded by plasmids DNA in vivo were detected by reverse transcription-polymerase chain reaction (RT-PCR) and western blot assay. Results indicated that all DNA vaccines could be well transcripted and expressed in injected tissues.
Two-week-old chickens were inoculated with DNA vaccines pVAX-LDH, pVAX-LDH-IFN-γ, pVAX-LDH-IL-2, recombinant LDH protein and inclusion bodies by leg intramuscular injection, respectively. A booster immunization was given 1 week later. Challenged control group and unchallenged control chickens were injected with sterile TE buffer at the same injection site. pVAX1 plasmid alone was given to chickens as plasmid control. All chickens except the unchallenged control group were challenged orally with 1.2×105 sporulated oocysts of E. acervulina 7 days following second injection. All chickens were euthanized to determine the effects of immunization on the 6th day post-challenge. The efficacy of immunization was evaluated on the basis of survival rate, lesion score, body weight gain, oocyst decrease ratio and anti-coccidial index (ACI). The results indicated the DNA vaccines could protect chickens from E. acervulina infection and pVAX-LDH-IFN-γand pVAX-LDH-IL-2 could induce better protection.
Two-week-old chickens were inoculated with DNA vaccines pVAX-LDH, pVAX-LDH-IFN-γ, pVAX-LDH-IL-2, respectively. A booster immunization was given 1 week later. Plasmid control group chickens and water control group were given pVAXl plasmid alone and sterile water, respectively. On day 7 post-second immunization, spleens were removed from five chickens and lymphocytes were prepared. Lymphocyte proliferation responses were determined by MTT. The results showed that splenocytes from DNA vaccinated chickens displayed significantly greater proliferation compared with plasmid control group chickens and water control group. This indicated that DNA vaccines constructed in this study could stimulate the immune system especially cell-mediated response of chickens.
Two-week-old chickens were inoculated with DNA vaccines pVAX-LDH, pVAX-LDH-IFN-γ, pVAX-LDH-IL-2, respectively. A booster immunization was given 1 week later. Plasmid control group chickens and water control group were given pVAX1 plasmid alone and sterile water, respectively. On day 7 and 14 post primary immunization, spleens and cecal tonsils were removed from five chickens of each group. Splenocytes were dually stained with mouse anti-chicken CD3+, CD4+ and CD8+ monoclonal antibody. Percentage of CD3+,CD4+/CD3+ and CD8+/CD3+ were determined by flow cytometric analysis. The results showed that percentage of CD3+T lymphocytes in lymphocytes obtained from spleen and cecal tonsil 7 days post primary injection showed no differences (p>0.05). However, on day 7 following booster, percentages of CD3+T cells of vaccinated groups in both spleen and cecal tonsil were significantly higher than that of control groups (p<0.05). This indicated that DNA vaccines constructed in this study could enhance T lymphocytes response of chickens.
Two-week-old chickens were inoculated with DNA vaccines pVAX-LDH, pVAX-LDH-IFN-γ, pVAX-LDH-IL-2, respectively. A booster immunization was given 1 week later. Plasmid control group chickens and water control group were given pVAX1 plasmid alone and sterile water, respectively. On day 7 post-second immunization, spleens and cecal tonsils were removed from chickens and lymphocytes were prepared. Total RNA was extracted from lymphocytes and cDNA was synthesized. The changes of IFN-γ, IL-2, IL-4, TNFSF15, IL-17D, TGF-β4 mRNA were determined by real-time PCR. The relative quantification of cytokine gene mRNA compared with the internal control gene GAPDH was calculated using 2-ΔΔCT method. The results showed that the levels of IFN-γ, IL-2, TNFSF15, IL-17D and TGF-β4 mRNA in spleens and cecal tonsils of chickens were significantly higher following DNA vaccination. This indicated that DNA vaccines constructed in this study could promote the secretion of cytokines.
Two-week-old chickens were inoculated with DNA vaccines pVAX-LDH, pVAX-LDH-IFN-γ, pVAX-LDH-IL-2, respectively. A booster immunization was given 1 week later. Plasmid control group chickens and water control group were given pVAX1 plasmid alone and sterile water, respectively. Whole blood from chicks was collected by cardiac puncture at weeks 1,2,3,4,5 and 6 post-second immunization and the serum was collected. The serum IgG antibody levels were determined by ELISA and E. acervulina LDH recombinant protein was used as coating antigen. The results showed that anti-LDH antibody titers were higher in chickens vaccinated with DNA vaccines compared to those of water control and plasmid control chickens during weeks 1-6 post-second immunization (P <0.05). Antibody titers were significantly greater in groups vaccinated with pVAX-LDH-IFN-γand pVAX-LDH-IL-2 than group immunized with pVAX-LDH (P<0.05). Chickens vaccinated with pVAX-LDH-IL-2 developed the highest antibody titer compared with the other two vaccinated groups. Non-specific antibody was detected in water control and plasmid control chickens throughout the experiment. These results indicated that DNA vaccines could induce specific antibody in chickens.
The results of immune protective effects and mechanism of DNA vaccines on chickens showed that DNA vaccines could simulate the systemic and local immune system of chickens and induce both cell-mediated and humoral responses to resist coccidiosis.
引文
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