白细胞介素2对固始鸡十二指肠黏膜免疫增强作用

以父母代固始鸡为试验材料,用添加不同剂量的白细胞介素2(IL-2)佐剂的鸡志贺氏菌苗对试验鸡进行分组免疫,应用免疫组织化学技术,通过Owin图像处理系统对鸡十二指肠内分泌型免疫球蛋白A(SIgA)阳性细胞反其分泌物的分布和阳性面积进行系统分析.结果表明,SIgA阳性细胞主要分布于十二指肠肠黏膜的固有膜及肠腺腔中,在志贺菌组中添加IL-2能够提高SIgA细胞的分泌,从而提高动物体的免疫能力,且以IL-2的添加剂量在50μg时对增加志贺菌苗的免疫性效果最佳.     

鸡Akirin2和GM-CSF促进传染性法氏囊病病毒VP2基因DNA疫苗免疫效果的研究

为提高鸡传染性法氏囊病病毒VP2基因DNA疫苗的免疫效力,将IBDV VP2、鸡Akirin2和鸡GM-CSF三基因编码区串联后克隆到真核表达载体pTriEx-4上,用构建的pT-VP2-Akirin2-GM-CSF重组质粒体外转染293T细胞,然后提取表达产物进行SDS-PAGE分析。结果在58、35、22ku附近各有一蛋白条带。用pT-VP2-Akirin2-GM-CSF免疫SPF鸡,加强免疫2周后,攻IBDV BC6-85强毒,4d后统计保护率。结果发现,注射14d后,pT-VP2-Akirin2-GM-CSF质粒免疫组血清中能检测到特异性IBDV抗体和IBDV中和抗体,第28天,该组鸡的抗体滴度极显著高于空白对照组(P0.01),该组鸡外周血淋巴细胞增殖能力也极显著高于空白对照组(P0.01)。说明VP2-Akirin2-GM-CSF三基因串联DNA疫苗有很好的应用前景。     

H7亚型禽流感病毒血凝素基因重组鸡痘病毒活载体疫苗免疫原性评估

本研究以细胞感染-转染技术构建了一株表达H7亚型禽流感病毒(AⅣ)血凝素(HA)基因的重组鸡痘病毒rFPV-HA.以5×105PFU的rFPV-HA经翅下刺种免疫8周龄SPF鸡,免疫后第3周用100 CID5o的HA基因同源AⅣ A/Arfi St/eng-Q/983/79(H7N1)进行人工感染,1周后采集泄殖腔棉拭子进行病毒分离.免疫后第三周及攻毒后第1、2周对所有动物进行静脉采血,检测血凝抑制(HI)抗体和免疫沉淀(AGP)抗体.结果,攻毒前部分rFPV-HA免疫鸡可检测到HI抗体,但检测不到AGP抗体,野生型病毒(F017)接种组和空白对照组HI抗体和AGP抗体均为阴性.攻毒后rFPV-HA免疫组的HI抗体上升速度明显高于野生型病毒接种组和空白对照组.rFPV-HA免疫组有6/8的鸡为流感病毒分离阴性,野生型病毒接种组和空白对照组的试验鸡病毒分离全部为阳性.结果表明rFPV-HA可诱导鸡产生有效的免疫保护反应,阻止或降低消化道病毒排泄.rFPV-HA的成功构建为研制开发H7亚型禽流感活载体疫苗奠定了良好的基础.     

禽流感灭活疫苗(H5N2,N28株)和重组禽流感灭活疫苗(H5N1,Re-1株)免疫鸡和鸭后抗体消长规律的比较研究

禽流感灭活疫苗（H5N2，N28株）和重组禽流感灭活疫苗（H5N1，Re-1株）各3批，以重组禽流感灭活疫苗（H5N1，Re．1株）推荐的免疫程序和剂量分组免疫5周龄SPF鸡和5周龄非免疫鸭。免疫后定期采集血样，测定血清HI抗体效价，分别计算各免疫组鸡和鸭HI抗体效价的几何平均数，绘制各批疫苗免疫鸡和鸭后HI效价消长曲线，并分别对两种疫苗免疫鸡和鸭后各时期的HI抗体效价进行t检验。结果为：①免疫后1周，各免疫组均未测到HI抗体；②鸡免疫后4～5周，HI抗体效价达到峰值1：2^6.0～1：2^7.0，随后2周下降相对较快，平均每周下降0．3—0．7个滴度，之后HI抗体效价缓慢下降，直到试验结束，平均每周下降0．04～0．17个滴度；③鸭首次免疫后第3周各免疫组HI抗体效价达到1：2^4.0～1：2^5.3，加强免疫（首次免疫后第3周）后，HI抗体效价迅速升高。加强免疫后第3周（首次免疫后第6周）HI抗体效价达到最高值1：2^10.6-1：2^11.7，且在峰值水平维持3周，之后1周下降相对较快，平均下降1．2～2．7个滴度，然后缓慢下降，到首免后24周平均每周下降0．28～0．39个滴度，24周除049H5N2批疫苗免疫组外，其他各批免疫鸡HI抗体效价均小于1：2^4.0；④免疫后3周内，两种疫苗刺激鸡产生的HI抗体效价均高于相应批次疫苗免疫鸭产生的HI抗体效价，免疫后第2周相差1．9—2．3个滴度，免疫后第3周相差1．6～2．0个滴度；⑤两种疫苗免疫鸡和鸭后，各时期HI抗体效价t检验差异均不显著（P〉0．05）。以上结果表明，两种疫苗免疫SPF鸡和非免疫鸭后，HI抗体效价消长规律一致，两种疫苗免疫后各个时期的HI抗体效价在统计学上差异均不显著（P〉0．05）。     

重组鸡IL-2对禽流感灭活苗免疫增强作用试验

使用重组鸡白介素-2作为免疫佐剂，检测chIL-2对禽流感病毒H9亚型灭活疫苗的免疫增强作用。将1日龄的AA肉雏鸡，随机分为7组，每组10只，采用胸部肌肉注射方式进行免疫，对照组仅接种H9亚型禽流感灭活疫苗，试验组分别同时接种H9亚型禽流感灭活疫苗O．5mL和不同剂量（0．01mg-1mg）的鸡IL-2。每7d静脉采血，采用血凝和血凝抑制方法检测机体中抗体水平变化情况。结果表明，接种重组鸡IL-2的试验组动物能在2月内保持高抗体水平，且在免疫后第10周，其平均抗体水平仍高于疫苗对照组3．6个～4．5个滴度，表明了重组鸡IL-2蛋白对H9亚型禽流感灭活疫苗具有免疫增强作用。     

重组白细胞介素2对固始鸡空肠黏膜免疫增强作用的研究

本试验旨在研究重组白细胞介素-2（interleukin-2,IL-2）对固始鸡空肠黏膜免疫的作用。 以父母代固始鸡为试验动物,用添加不同剂量的重组IL-2佐剂的鸡新城疫疫苗对试验鸡进行分组免疫,应用免疫组织化学技术,通过Qwin图像处理系统对鸡空肠内分泌型免疫球蛋白A（sIgA）阳性细胞及其分泌物分布和阳性面积量进行系统分析。结果表明,sIgA阳性细胞及其分泌物主要分布于肠绒毛的固有层和肠腺腔中。在新城疫疫苗组中添加重组IL-2能够提高sIgA细胞的分泌,从而提高动物体的免疫能力。重组IL-2对增强免疫性有一定的作用,且IL-2的添加剂量在50 μg时对增加新城疫疫苗的免疫性效果最佳。      

山羊免疫弓形虫代谢分泌抗原后的免疫应答

将山羊随机分为6组，即E／SA纽、E／SA＋cpG组（未乳化）、E／SA＋CpG组、E／SA＋IL-2组、E／SA＋IL-2＋CpG组、对照组，每组3只，分别于免疫前、免疫后第2周、免疫后第4周、感染后第1周及感染后第2周采集山羊外周全血及涂血片，IHA法检测抗体滴度的动态变化；ELISA法检测IFN-γ、TNF—a、IL-2、IL-4的表达水平；ANAE染色法检测T淋巴细胞的动态变化。结果显示，免疫后各免疫组的IFN-γ、TNF-a、IL-2、IL-4及T淋巴细胞水平显著高于对照组（P〈0．05），各免疫组的抗体水平均高于对照组。结果表明，E／SA可引起IFN—γ、TNF—a、IL-2、IL-4、T淋巴细胞及抗体水平的升高，说明E／SA免疫后可引起山羊较强的细胞免疫和体液免疫应答。     

禽流感DNA疫苗和转移因子联合免疫效果研究

为研究禽流感DNA疫苗和转移因子联合免疫的免疫增强效果,试验用DH5α工程菌增殖的重组质粒pCAGGoptiHA5和转移因子联合免疫SPF鸡(转移因子采用口服和注射两种方式进行协同免疫),免疫后不同时间监测各组免疫鸡只的免疫器官指数、血清HI抗体水平变化情况。结果表明:联合免疫试验组抗体水平显著高于DNA疫苗单独免疫组,联合免疫组鸡只的法氏囊、胸腺和脾脏免疫器官指数均显著增加(P0.05),但转移因子的口服和注射两种免疫方式之间差异不显著(P0.05)。说明禽流感DNA疫苗和转移因子联合免疫协同作用显著。     

重组鸡α-干扰素对鸡新城疫感染的治疗及疫苗免疫的影响

对试验鸡进行新城疫人工感染的干扰素治疗试验,试验SPF鸡分成5组,1～3组分别于攻毒前24h、攻毒的同时和攻毒后24h注射重组鸡α-干扰素,4、5组作为病毒对照和空白对照。同时,还进行了重组鸡α-干扰素对新城疫弱毒苗和灭活苗免疫效果的影响试验,结果显示,试验2、3组的平均死亡时间明显延长,免疫试验免疫后14d注射重组鸡α-干扰素可以降低新城疫抗体的滴度。     

溶菌酶对肉鸡免疫机能的影响

试验旨在探讨溶菌酶对内鸡免疫机能的影响.试验选取144只1日龄艾维因肉鸡,随机分为4个处理组:对照组、抗生素组及2个剂量溶菌酶组,每组6个重复,每重复6只鸡.42日龄取样,计算免疫器官指数,检测血清新城疫抗体水平和脾细胞因子相对表达水平.试验结果表明:溶菌酶和抗生素在一定程度上降低肉鸡免疫器官指数和抗体水平,有降低动物免疫活化状况的趋势;溶菌酶组2处理可极显著降低脾IL-1β mRNA水平,不同试验处理组的脾TNF-α mRNA水平均有所降低,但差异不显著.     

钩吻对固始鸡生长性能及免疫性能的影响

通过在3周龄固始鸡饲粮中添加不同剂量（0.4%、0.8%、1.2%、1.6%）的钩吻（GEB）,测定了其对固始鸡生长性能和免疫性能的影响.添加GEB后,日增重、料肉比有所提高,但差异不显著（P〉0.05）,其中添加0.8%GEB日粮组对固始鸡的日增重影响最明显,料肉比最低;添加GEB后,固始鸡的胸腺、脾脏、法氏囊指数均比对照组高,但差异不显著（P〉0.05）,添加一定剂量GEB后,固始鸡血清中针对新城疫病毒（NDV）的抗体效价均比对照组高,5周龄和7周龄时0.8%GEB组、1.2%GEB组和1.6%GEB组显著高于对照组（P〈0.05）,9周龄的NDV抗体效价高于对照组但差异不显著（P〉0.05）.     

Study on the Activity Estimation Methods of Recombinant Chicken Interferon-α/Interleukin-2 Fusion Protein in vitro

In order to establish a stable,simple and rapid detection method to estimate the activities of the recombinant chicken interferon-α/interleukin-2 fusion protein (rChIFN-α-Linker-ChIL-2,recombinant fusion protein) in vitro,the activities of rChIFN-α-Linker-ChIL-2 were estimated by detecting its specific immune response to monoclonal antibody (MAb) against ChIFN-α and ChIL-2 by ELISA assay.The antiviral activities of rChIFN-α-Linker-ChIL-2 protein were tested by inhibiting the 50% appearance of cytopathic effect (CPE) of vesicular stomatitis virus (VSV) and infectious bursal disease virus (IBDV) on the passage cell lines DF1.The promoting proliferation activities of lymphocytes in the chicken peripheral blood and spleen of recombinant fusion protein were tested by MTS method.The results showed that rChIFN-α-Linker-ChIL-2 protein had the ability of specific immune response to anti-ChIFN-α MAb and anti-ChIL-2 MAb,respectively.The antiviral activity of rChIFN-α-Linker-ChIL-2 protein inhibiting the reproduction of VSV on DF1 cell line was higher than IBDV,and both of the antiviral activities of recombinant fusion protein against VSV and IBDV were much higher than the recombinant ChIFN-α protein (rChIFN-α) control.The recombinant fusion protein had apparent promoting proliferation activity of lymphocytes in the chicken peripheral blood and spleen,which were much higher than that of the rChIFN-α control.The study suggested that the activities detection and estimation methods of the recombinant fusion protein in vitro were successfully established,which laid the foundation for the further study of the synergy activity of recombinant fusion protein in vivo.     

重组鸡α干扰素/白细胞介素-2融合蛋白体外活性评价方法研究

为建立稳定、便捷的重组鸡α干扰素/白细胞介素-2融合蛋白(rChIFN-α-Linker-ChIL-2蛋白,重组融合蛋白)体外活性评价方法,本研究分别采用ChIFN-α和ChIL-2 ELISA方法检测重组融合蛋白与抗ChIFN-α单抗和抗ChIL-2单抗发生特异性免疫反应的活性;采用细胞病变抑制法检测重组融合蛋白在DF1细胞上抑制水疱性口炎病毒(VSV)和传染性法氏囊病病毒(IBDV)增殖活性;采用MTS法分别测定重组融合蛋白促鸡外周血T淋巴细胞(PBLC)和脾淋巴细胞增殖活性。结果表明,重组融合蛋白可以与抗ChIFN-α单抗和抗ChIL-2单抗发生特异性免疫反应;重组融合蛋白在DF1细胞上具有明显抗病毒活性,其抗VSV活性高于抗IBDV活性,且均明显高于rChIFN-α蛋白对照;不同浓度的重组融合蛋白均具有明显的促鸡PBLC和脾淋巴细胞增殖活性,且其促增殖活性明显高于rChIFN-α蛋白对照。本研究成功建立了重组融合蛋白体外活性检测评价方法,为进一步探究重组融合蛋白在鸡体内协同作用奠定基础。     

Effect of Different Rearing Conditions of Indoor and Outdoor in Winter and Summer on Growth Performance and Immune Function of Heijiao MA Chicken

This experiment was conducted to study the effect of different rearing conditions of indoor and outdoor in winter and summer on growth performance and immune function of Heijiao MA chicken.In the winter and summer, 180 Heijiao MA chicken with the age of 35-day-old were chosen and randomly divided into two groups with three replicates per group and 30 chickens per replicate.The Heijiao MA chicken were reared at outdoor and indoor, respectively, and at 49, 63, 77, 91 days the immune organ indexes, serum antibody titers and growth performance were measured.The results showed that:①Whether it was winter or summer, the body weight of Heijiao MA chicken rearing at indoor were higher than that of outdoor, and the difference was significant at experimental stages in winter (P<0.05).There was significant difference only at 77 days old of Heijiao MA chicken in summer (P<0.05), the rest were not significant (P>0.05);②The ADG of outdoor group in summer was higher than that in winter, while the F/G of outdoor group in summer were significantly lower than that in winter (P<0.05).The ADFI and ADG of indoor group were not significantly different between winter and summer (P>0.05).③On immune function, immune organ indexes and ND antibody titer of Heijiao MA chicken of indoor group in winter were higher than outdoor group, while part of immune organ indexes, AI (H5) and ND antibody titers of outdoor group in summer were higher than that of indoor group;And ND and AI (H5) antibody titers in winter were higher than summer.In conclusion, The climate and season were major factors to influence the growth performance and immune function of Heijiao MA chicken.     

冬夏两季室内、外饲养对黑脚麻鸡生长性能和免疫功能的影响

为探究不同季节室内、外饲养方式对黑脚麻鸡生长性能和免疫功能的影响,本试验在冬季、夏季各选用180只35日龄的黑脚麻鸡雌雏,随机分成2组,每组3个重复,每个重复30只,分别进行室外放养和室内平养。在49、63、77、91日龄分别对黑脚麻鸡生长性能、免疫器官指数和血清抗体效价进行测定。结果显示,①无论是冬季还是夏季,试验期室内平养的黑脚麻鸡体重均大于室外放养,冬季两试验组均差异显著(P<0.05),夏季仅在77日龄时差异显著(P<0.05),其余日龄差异均不显著(P>0.05);②夏季室外放养组黑脚麻鸡平均日增重(ADG)高于冬季室外放养组,料重比(F/G)显著低于冬季室外放养组(P<0.05),而室内平养组平均日采食量(ADFI)和ADG在两个季节间无显著差异(P>0.05);③在免疫功能上,冬季室内平养黑脚麻鸡免疫器官指数和新城疫(ND)抗体效价均大于室外放养组,而夏季室外放养组部分免疫器官指数和禽流感H5亚型(AI(H5))、ND抗体效价高于室内平养组;冬季两种抗体效价均高于夏季。综上所述,气候和季节是影响放养黑脚麻鸡生长性能及免疫功能的主要因素。     

The Influence for Chickens' T Lymphocyte Subsets in Peripheral Blood after Inoculated with the Recombinant Chicken Interferon-α/Chicken Interleukin-2 Complex Protein

To explore the influence of recombinant chicken interferon-α/chicken interleukin-2 fusion protein (rChIFN-α-Linker-ChIL-2 protein, recombinant fusion protein) on the percentage content of peripheral T lymphocyte subsets (PBLC) of SPF chicken,the flow cytometry was used to detected the percentage contents of CD3⁺CD4⁺, CD3⁺CD8⁺ and the CD4⁺/CD8⁺ ratio in PBLC at different days post injection in the 14-day-old SPF chicken injected with rChIFN-α-Linker-ChIL-2 protein (group 2) and recombinant chicken interferon α (rChIFN-α) protein (group 3). The results indicated that the recombinant fusion protein and rChIFN-α protein could increase the percentage content of CD3⁺CD4⁺ T lymphocyte, decrease the percentage content of CD3⁺CD8⁺ T lymphocyte, improve CD4⁺ /CD8⁺ T lymphocyte ratio during 3 to 14 d post injection. The percentage contents of CD3⁺CD4⁺, CD3⁺CD8⁺ and CD4⁺/CD8⁺ ratio in chickens of group 2 were significantly different from the PBS control group (group 1) (P<0.01), and were notably different from group 3 during 3 to 7 d post injection (P<0.05). These results showed that both the rChIFN-α-Linker-ChIL-2 protein and rChIFN-α protein could affect the percentage contents of lymphocyte subsets, improve CD4⁺/CD8⁺ ratio, enhance the cellular immune function in chicken. The influences of rChIFN-α-Linker-ChIL-2 protein on the percentage contents of CD3⁺CD4⁺ and CD3⁺CD8⁺ T lymphocyte, and CD4⁺/CD8⁺ value were notable higher than rChIFN-α, which suggested that recombinant fusion protein play a synergistic role of interferon alpha and interleukin-2 on the cellular immune function by this pathway in chicken.     

糖萜素对固始鸡免疫器官发育及T淋巴细胞亚群的影响

应用流式细胞仪技术检测固始鸡CD3⁺、CD4⁺和CD8⁺ T淋巴细胞含量来研究添加不同剂量的糖萜素对固始鸡免疫器官及T淋巴细胞亚群的影响。结果表明,500、700 mg/kg糖萜素组的免疫器官指数及CD4⁺／CD8⁺比值显著(P<0.05)高于其余各组。因此,一定剂量的糖萜素可以促进固始鸡免疫器官发育,提高T淋巴细胞亚群的水平。     

鸡α-干扰素对罗曼雏鸡免疫功能的影响

为研究鸡α-干扰素对罗曼雏鸡免疫功能的影响,将60羽健康1日龄罗曼雏鸡随机分为2组,即对照组、试验组。试验组鸡于1日龄胸肌注射鸡-α干扰素,0.5 mL/羽;对照组鸡同期每羽注射生理盐水0.5 mL。试验开始后,于第4 d、8 d、12 d、16 d、20 d对鸡进行翅下静脉采血,并于20日龄剖杀摘取免疫器官。研究表明,鸡注射α-干扰素后,其外周血液ANAE+T淋巴细胞百分率和鸡脾相对湿重显著高于对照组,鸡α-干扰素在一定范围内可提高雏鸡免疫功能。