蜂胶作为动物疫苗佐剂的研究及应用

<正>蜂胶(Propolis)是一种具有芳香气味的胶状固体物,由蜜蜂从树干或植物芽孢上采集树脂(树胶)后,与其上腭腺、蜡腺自身分泌的的分泌物混合加工而成。其内含有丰富的生物活性物质,具有多种生物学特性,兼具广谱抗病毒、抗细菌、抗霉菌、消炎、止痒、增强免疫、抗氧化、降血糖、降血脂等多种功能,广泛应用于人类的医疗、保健行业,作为新兴的保健品也倍受推崇,现已成为各国科学研究的热点[1]。蜂胶用作疫苗佐剂优势明显,获得了良好效果,迅速成为了当前佐剂开发热潮中的佼佼者,被广泛应用于动物疫苗领域,并     

重组大肠杆菌肠毒素蛋白LTA_(192)-STa_(13)的表达及其诱导小鼠体液免疫的研究

为研究重组大肠杆菌肠毒素融合蛋白LTA192-STa13的免疫原性,本研究将产肠毒素大肠杆菌(ETEC)elt A和est A基因定点突变后构建融合基因,进行重组蛋白LTAR192G-STaG13Q的表达。利用纯化的重组蛋白和灭活菌液分别免疫小鼠,比较不同抗原诱导小鼠体液免疫应答能力,以及体外免疫血清对毒素的中和作用。结果显示,各组免疫小鼠均可以产生高滴度抗LTA和STa抗体,其血清抗体可以通过乳汁传递给新生仔鼠,其中蛋白免疫组血清抗体效价更高。体内外中和试验证明,实验组小鼠三免后的血清能够中和LT和STa毒素对实验鼠的毒性作用,并且能够中和LT对Y-1细胞的毒性作用,蛋白免疫组血清抗体中和效价可达到1∶89.13,明显高于灭活菌免疫组。研究表明,重组蛋白LTA192-STa13对小鼠具有良好的免疫原性,能够诱导免疫鼠产生良好的体液免疫应答,可以作为ETEC亚单位疫苗的候选抗原。     

产气荚膜梭菌α-β2-ε毒素融合蛋白在大肠杆菌中的表达及免疫原性研究

为研究产气荚膜梭菌α、β、ε3种主要外毒素的免疫原性,构建基因亚单位多价疫苗,本研究通过PCR分别扩增α-β2及ε3种毒素基因,分别克隆于p Pro HTa载体中构建重组表达质粒p Pro-α-β2-ε,并转化大肠杆菌进行这3个目的基因的融合表达。SDS-PAGE分析显示,表达的α-β2-ε毒素融合蛋白大小约90 ku,主要以包涵体形式存在。将其免疫BALB/c小鼠后,分别利用A型和B型产气荚膜梭菌强毒素对免疫鼠进行攻毒,并测定免疫鼠血清中抗体对毒素的中和活性。结果表明,免疫鼠对A型和B型产气荚膜梭菌毒素1 LD100和2 LD100的攻毒保护率分别为100%和60%以及100%和80%。体外中和试验显示,免疫鼠血清稀释为1∶20时,对1 LD100A型和B型产气荚膜梭菌毒素的中和效率均可达到100%。以上结果表明,本研究制备的α-β2-ε融合蛋白具有良好的免疫原性,能够有效刺激机体产生中和抗体,可以作为基因工程疫苗的有效组分,为产气荚膜梭菌多价候选亚单位疫苗的研究奠定了基础。     

TNF-α基因在F18大肠杆菌抗性型和敏感型断奶仔猪间的差异表达分析

本研究运用Real-time PCR方法分析TNF-α基因在苏太猪F18大肠杆菌抗性型和敏感型断奶仔猪群体中11个组织中的表达谱,以及在抗性型和敏感型个体之间的差异表达,以探讨TNF-α基因在断奶仔猪抗F18大肠杆菌感染中的作用。结果表明:TNF-α基因在抗性型和敏感型个体所检测的11个组织中均有表达,其中在脾脏、肺、肾脏、胸腺和淋巴结组织中表达量较高,在十二指肠和空肠组织中呈中度表达;TNF-α基因在F18大肠杆菌抗性型个体各个组织中的表达量普遍高于敏感型个体,且脾脏、肾脏、胸腺和十二指肠组织中TNF-α基因在抗性型个体中的表达量显著高于敏感型个体(P0.05)。由此推测,TNF-α基因的较高表达可能有利于提升仔猪对F18大肠杆菌的抵抗能力,在F18大肠杆菌侵袭后引起的一系列免疫应答过程中发挥着重要的作用。     

产后奶牛子宫内大肠杆菌的分离和血清型鉴定

分析产后健康和子宫内膜炎奶牛子宫内大肠杆菌的分布和血清型与患病之间的关系。选取产后21~30 d的健康奶牛25头和子宫内膜炎奶牛35头,采集子宫黏液,常规方法对分离的菌株进行生化鉴定和O抗原血清型分型。结果显示,60头奶牛中43头(71.67%)分离到145株大肠杆菌,61株来自18头健康牛,84株来自患子宫内膜炎奶牛,说明大肠杆菌在产后21~30 d奶牛子宫内的分离率较高。血清学分型发现,96株菌分属于不同的15种血清型,3株发生自溶,46株未能确定,87.5%(84株)的菌株属于7种常见血清型,即O2、O5、O26、O44、O73、O86和O111,而O111(41.84%)和O44(27.55%)是最为集中的两种类型,O2,O8、O55和O86仅出现在患病奶牛中,O114、O126、O141仅出现在健康奶牛中。说明一些特异性的血清型可能与患病有关。     

Study on Effect of Nitrogen and Phosphorous on the Resistance of E.coli to Chloramphenicol and its Mechanism

This study was designed to explore the effect of nitrogen and phosphorous on the resistance of E.coli from environment and the mechanism.Microcosms were established to study the effect of nitrogen and phosphorous on the resistant phenotype of E.coli to chloramphenicol (CHL).cat gene of isolated drug-resistant strains and susceptible strains were detected.The results showed that,different concentration of nitrogen and phosphorous could induce the formation of antibiotics resistance of E.coli to CHL.The rate of cat gene of 46 strains of chloramphenicol resistant E.coli was 89.13%,which was 0 in the 16 strains of chloramphenicol sensitive E.coli.The results indicated that,nitrogen and phosphorous in the microcosms could induce the formation and maintenance of resistance to chloramphenicol in E.coli,which had correlation with cat gene.     

Construction and Identification of PRRSV ORF5 Gene Prokaryotic Expression Vector

The present study was designed to construct recombinant plasmids,which could express porcine reproductive and respiratory syndrome virus (PRRSV) ORF5 gene.RNA was extracted from spleen and lung samples of the suspected pigs which were infected with PRRSV.According to PRRSV ORF5 gene,a pair of primers was designed for RT-PCR amplification.The ORF5 target gene was cloned into pMD19-T vector and then the recombinant pMD19-ORF5 was achieved.According to the sequencing results and the characteristics of expression vectors,a pair of primers with NcoⅠand XbaⅠenzyme cleavage sites was designed.Target fragment dORF5 was amplified and then connected to pProEXHTb and pNZ8149 vectors,respectively.And recombinant HTb-dORF5/DE3 and pNZ8149-dORF5/NZ3900 was induced by IPTG and Nisin,respectively,and analyzed by SDS-PAGE and Western blotting.Recombinant HTb-dORF5/DE3 induced by 1.5 mmol/L IPTG was expressed in the highest quantity.There were specific band at about 22 ku with reactionogenicity when it was tested by SDS-PAGE and Western blotting.Recombinant pNZ8149-dORF5/NZ3900 induced by 20 ng/mL Nisin was expressed in the highest quantity.There were specific band at about 19 ku with reactionogenicity when it was tested by SDS-PAGE and Western blotting.The IFA result showed specific green fluorescence.This study successfully constructed recombinant plasmids HTb-dORF5 and pNZ8149-dORF5 and expressed,the result laid a solid foundation for further development of PRRS vaccines.     

Study on Drug Resistance of Escherichia coli Isolated from Chicken and Swine in Organic Farm

To determine the drug resistance of E.coli strains from swine and chicken in organic farm,collection of stool specimens was conducted in one organic farm in Conghua city,Guangdong province in 2014,in which 118 E.coli strains were separated,including 55 from chicken and 63 from swine.The sensitivity of the E.coli strains from swine and chicken to 18 kinds of drugs was detected through agar dilution method.Similar properties in drug resistance were observed both in E.coli strains from swine and chicken in the same organic farm.In consideration of the resistance rate,samples were relatively sensitive to ceftazidime,cefquinome,cefoxitin,amikacin,apramycin,gentamicin,neomycin,florfenicol,chloramphenicol,imipenem,ciprofloxacin and olaquindox (chicken 0 to 21.8%;swine 0 to 14.3%).These isolates showed moderate resistance to ampicillin and streptomycin (chicken 29.1% to 38.2%;swine 23.8% to 27.0%).About 56.4% chicken isolates and 47.6% swine isolates showed resistant to more than 3 kinds of drugs.However,severe resistance was observed in trimethoprim-sulphamethoxazole,tetracyclineand and doxycycline (chicken 45.5%,76.4% and 72.7%;swine 50.8%,81.0% and 68.3%).The results also indicated that under organic farming mode,the drug resistance rates of E.coli strains from swine and chicken were relatively low in general.Multi-drug resistance existed but was relatively slight.Although the drug resistance rate to several specific antimicrobials was high,it was still lower than that in conventional farms.Since most studies focused on the drug resistance of the bacteria under conventional farming model,this study filled the blank of the research of drug resistance under organic farming model.     

不同寡糖对肉仔鸡肠道微生物的影响

本试验研究了4种寡糖对健康艾维茵肉仔鸡肠道微生态的影响.选用健康艾维茵肉仔鸡20只,随机分为5个处理,每个处理4个重复,每个重复1只鸡,进行为期40 d的饲养试验.试验第1组为对照组,饲喂基础日粮,第2、3、4、5组为基础日粮中分别添加同一水平糖量(0.015％)的低聚异麦芽糖组(GOS)、低聚木糖组(XOs)、果寡糖组(FOS)、甘露寡糖组(MOS).饲喂40 d后,研究寡糖对内仔鸡盲肠微生物(乳酸杆菌、双歧杆菌、大肠杆菌)数量的影响.结果表明,各种低聚糖能显著降低肉仔鸡盲肠微生物中大肠杆菌的数量(P＜0.05),其中XOS能显著提高盲肠微生物中双歧杆菌与大肠杆菌的比值,显示出良好的促进双歧杆菌增值的作用;MOS能显著提高盲肠微生物中乳酸杆菌与大肠杆菌的比值,显示出良好的促进乳酸杆菌增值的作用.     

肉鸡大肠杆菌病的调查和诊治

随着如东地区个体肉鸡养殖业的发展,肉鸡的大肠杆菌病的发病率和死亡率逐步增高,已成为危害该县的肉鸡生产的重要疾病之一。2013年4月初到5月中旬对该县及周边地区的乡镇的肉鸡发生的大肠杆菌病进行调查和研究,详细记录饲养状况和发病情况,选择了大量病料进行细菌分离培养,革兰氏染色镜检,并做一些生化实验进行确诊。此外,根据大肠杆菌容易产生耐药性的特点,我们做了大量的药敏实验,选择该地区病原比较敏感的富络欣（氟苯尼考）、利可宁（硫酸粘杆菌素和甲氧苄啶）两种药物,联合用药进行治疗,取得了比较好的效果。