禽霍乱-新城疫二联油乳剂灭活疫苗研究 (Ⅳ报)——疫苗的田间试验

[目的]评价禽霍乱-新城疫二联油乳剂灭活疫苗的临床应用效果,为联合防制禽霍乱和新城疫提供条件。[方法]将禽A型多杀性巴氏杆菌1502强毒株与鸡新城疫病毒La Sota弱毒株混合,制备成5批合格的二联油乳剂灭活苗,用于鸡、鸭和鹅的田间安全性和免疫保护试验。[结果]田间安全性试验表明,免疫鸡、鸭和鹅均未出现不良反应;鸡的田间免疫效力试验表明,7～14日龄雏鸡和60～90日龄青年鸡免疫3周后新城疫血凝抑制（ND-HI）抗体效价均比对照组高2～3 log2,可持续4个月以上,禽多杀性巴氏杆菌攻毒保护率均达到75.0%以上,免疫效力可持续6个月;鸭和鹅的田间免疫效力试验表明,接种3周后免疫组ND-HI抗体效价均≥4.2 log2,对照组均≤2 log2;鸭、鹅的禽多杀性巴氏杆菌攻毒保护率分别在75.0%和62.5%以上。[结论]该二联苗免疫禽类安全可靠,具有良好的免疫效果。     

Immunization of Male Mice with a New Recombinant GnRH Fusion Protein Reduces the Testicular Function

The objective of the present study was to evaluate the effect of an immunogenic maltose-binding protein-gonadotropin releasing hormone （GnRH-6-MBP） using genetic engineering. The synthetic mammalian tandem repeated GnRH hexamer gene was inserted into the expression plasmid pMAL-c2x. Recombinant GnRH-6-MBP protein was over- expressed in E.coli strain BL21. Amylose resin with affinity chromatograph was used to purify target protein. The reactiongenicity of fusion protein was identified by indirect enzyme linked immunosorbent assay （ELISA）, and the antigenicity and biological effects of GnRH-6-MBP were tested in mice. In the experiment, 20 male Kunming white mice of 20 d old were randomly divided into treatment and control group. Ten mice were immunized with 100 μg GnRH-6-MBP administered subcutaneously （s.c.） thrice at 2-week intervals with GnRH-6-MBP. Mice were sacrificed after 3 weeks following the booster injection, the testis was removed, weighed and measured, and the histological structure was observed. The reactiongenicity of fusion protein to GnRH antibody was much higher than the control. Active immunization against GnRH-6-MBP reduced remarkably （P 〈 0.01） the length and weight of the testis, and shortened the girth and width of the testis （P 〈 0.05）, and suppressed testicular spermatogenesis compared to the control mice. These results indicate that the recombinant GnRH-6-MBP acted as a strong immunogen and caused atrophy of the testis.     

Evaluating the protective efficacy of antigen combinations against Photobacterium damselae ssp. piscicida infections in cobia,Rachycentron canadum L.

Cobia, Rachycentron canadum L., is a very important aquatic fish that faces the risk of infection with the bacterial pathogen Photobacterium damselae ssp. piscicida, and there are few protective approaches available that use multiple antigens. In the present study, potent bivalent antigens from P. damselae ssp. piscicida showed more efficient protection than did single antigens used in isolation. In preparations of three antigens that included recombinant heat shock protein 60 (rHSP60), recombinant α‐enolase (rENOLASE) and recombinant glyceraldehyde‐3‐phosphate dehydrogenase (rGAPDH), we analysed the doses that elicited the best immune responses and found that this occurred at a total of 30 μg of antigen per fish. Subsequently, vaccination of fish with rHSP60, rENOLASE and rGAPDH achieved 46.9, 52 and 25% relative per cent survival (RPS), respectively. In addition, bivalent subunit vaccines – combination I (rHSP60 + rENOLASE), combination II (rENOLASE + rGAPDH) and combination III (rHSP60 + rGAPDH) – were administered and the RPS in these groups (65.6, 64.0 and 48.4%, respectively), was higher than that achieved with single‐antigen administration. Finally, in combination IV, the trivalent vaccine rHSP60 + rENOLASE + rGAPDH, the RPS was 1.6%. Taken together, our results suggest that combinations of two antigens may achieve a better efficiency than monovalent or trivalent antigens, and this may provide new insights into pathogen prevention strategies.     

罗非鱼无乳链球菌C5a肽酶(ScpB)的原核表达及其免疫原性

以尼罗罗非鱼(Oreochromis niloticus)为鱼体样本, 克隆了罗非鱼源无乳链球菌(Streptococcus agalactiae) scpB基因, 构建了原核表达质粒pET32a(+)-scpB, 转入大肠杆菌BL21(DE3)后, IPTG诱导表达, 表达的重组蛋白经镍离子金属螯合柱纯化及超滤管浓缩后, 进行SDS-PAGE和Western blot分析鉴定。纯化的重组蛋白以不同剂量(1 μg/g、3 μg/g、5 μg/g) 免疫尼罗罗非鱼后, 每周检测各实验组鱼体血清非特异性免疫指标[溶菌酶活性、超氧化物歧化酶(SOD)活力]及抗体水平变化情况, 并于免疫4周后以4倍LD₅₀的剂量对其进行人工攻毒。结果显示, 该重组蛋白在大肠杆菌中以包涵体的形式存在; 对尼罗罗非鱼的相对免疫保护率为69.66%~89.00%, 其中5 μg/g组的相对保护率最高; 受免鱼体血清中溶菌酶活性、SOD活力和抗体水平较对照组有极显著提高(P<0.01), 结果表明, 重组蛋白ScpB具有较强的免疫原性和保护作用。本研究旨在为GBS多肽疫苗的研制奠定基础。

     

影响草鱼出血病疫苗免疫效果因素风险评估

 应用模糊层次分析法, 并结合流行病学调查、荟萃分析和德尔菲法, 构建了由评估指标体系、风险因素权重、评分标准、综合评价函数等组成的草鱼出血病免疫预防风险评估模型, 其中, 评估指标体系包括疫苗品质(B1)、免疫程序(B2)、鱼体(B3)、池塘环境(B4)和饲养管理(B5)共5个准则层风险因素和疫苗品种(C1)、保存温度与有效期(C2)、运输存储条件(C3)、免疫时疫苗的存放(C4)、免疫时鱼体健康状态(C5)、免疫技术(C6)、免疫剂量(C7)、漏免的鱼数(C8)、鱼种来源(C9)、健康状态(C10)、鱼体规格(C11)、水温(C12)、溶氧(C13)、氨氮(C14)、亚硝酸盐(C15)、pH值(C16)、透明度(C17)、水色(C18)、底泥厚度(C19)、载鱼量(C20)、搭配模式(C21)、药物的使用(C22)、饲料(C23)、青草投喂(C24)等24个指标层风险因素; 5个准则层风险因素权重值集合为W={0.267; 0.102; 0.131; 0.263; 0.237}, 24个指标层风险因素绝对权重集合为W={0.138;0.059;0.046; 0.024; 0.035; 0.018; 0.027; 0.022; 0.040; 0.054; 0.037; 0.076; 0.037; 0.032; 0.030; 0.027; 0.018; 0.019; 0.024; 0.104; 0.024; 0.027; 0.062; 0.020}; 分别建立了定性和定量评估指标的评分标准, 并以综合评价函数 表示风险评估结果。应用该模型评估了华南地区36个免疫池塘发病风险, 结果显示, 3个发病池塘风险值分别为0.572、0.638、0.617, 处于高度风险级别, 与未发病塘存在极显著差异(P<0.01), 评估结果较准确, 表明该模型可应用于草鱼出血病免疫预防管理和决策。

     

不同类型H5亚型禽流感疫苗的免疫保护效力比较研究

为比较以我国首个H5亚型禽流感病毒分离株A/Goose/Guangdong/1/1996(H5N1)\[GS/GD/96\]为基础构建的新型重组禽流感病毒灭活疫苗（H5N1亚型,Re\|1株）、禽流感重组鸡痘病毒载体活疫苗（H5亚型）、禽流感、新城疫重组二联活疫苗（rLH5\|1株）以及禽流感DNA疫苗（H5亚型,pH5\|GD）的免疫保护效力。分别将四种禽流感疫苗以2.8 μg HA/0.3 mL（肌肉注射）、103PFU/100 μL（刺种）、106EID50/100 μL（滴鼻、点眼）、15 μg/200 μL（肌肉注射）等不同剂量和方式免疫3周龄SPF鸡,首次免疫3周后再加强免疫一次,加强免疫2周后用106EID50的高致病力禽流感病毒（HPAIV） GS/GD/96鼻腔途径进行攻击,观察发病与死亡情况。分别于攻毒后第3 d、第5 d、第7 d采集喉头及泄殖腔拭子进行病毒分离、滴定检测排毒情况,同时检测免疫及攻毒后血清HI抗体的动态变化。所有疫苗均可对免疫鸡形成100%完全保护（不发病、不致死、不排毒）;在病毒攻击前,灭活疫苗（H5N1亚型,Re\|1株）、禽流感重组鸡痘病毒载体活疫苗（H5亚型）、禽流感、新城疫重组二联活疫苗（rLH5\|1株）以及禽流感DNA疫苗（H5亚型,pH5\|GD）所诱导的HI抗体平均水平分别为875log2、6.5log2、5.13log2和7.88log2。新型的基因工程疫苗具有良好的免疫保护性,已经或将在H5亚型HPAIV的防治实践中起到有益的补充作用。     

多表位疫苗的构建策略及其在动物疫苗中的应用

多表位疫苗是基于抗原表位氨基酸序列而制备的一种新型分子疫苗。由于具有安全性好、保护力强和应答类型可以有效调控等优点,多表位疫苗具有较高的研究和应用价值。多表位疫苗因其独特的免疫优势已成为近年来基因工程疫苗研究领域的热点之一。本文概述了表位的预测筛选、多表位疫苗的设计与优化及其在动物医学领域的应用等方面的进展。     

左旋咪唑对新城疫灭活疫苗抗体滴度的影响

为探讨免疫增强剂左旋咪唑对新城疫灭活疫苗抗体滴度的影响,将20只20日龄雏鸡,随机分成A、B、C、D4组,每组5只。分别皮下注射新城疫灭活疫苗0.5ml/羽,同时注射A组雏鸡高(0.6mg)、B组中(0.3mg)、C组低(0.15mg)剂量的左旋咪唑,对照组D不加。免疫后0天、7天、14天、21天和28天,翅下静脉采血并检测新城疫抗体滴度。结果显示:免疫前4组抗体水平基本一致,无统计学意义;免疫后7天,14天,21天,高、中、低3组抗体水平基本一致,4组的抗体水平相差均在2个滴度以内,统计分析无明显差异;免疫后28天,B组抗体水平较高。统计分析显示,对照组与高、中、低3组均有显著差异,但无剂量差异;在高、中、低3组中,以中剂量添加最佳,抗体效价高(7.2log2),但中剂量组与高、低组的差异不显著。这表明,左旋咪唑的添加提高新城疫抗体滴度,并且与免疫后时间长短有关系。从趋势看,随着时间的推移,添加组的抗体水平好于对照组。