减毒沙门氏菌为载体的草鱼口服生长抑素DNA疫苗的构建及其稳定性

将生长抑素(SS)和乙肝表面抗原基因(HBsAg)融合后，插入真核表达载体pcDNA3，构建成pcDNA3-SS，再转化减毒沙门氏菌(S．typhimurium,dam^-和phop^-)，构建了以减毒沙门氏菌为载体的生长抑素口服DNA疫苗ZJ111／pcDNA3-SS，并通过体外传代、灌服草鱼检验了ZJ111／pcDNA3-SS的侵袭力及体外和侵入草鱼体内过程中的德定性。对草鱼肝脏和脾脏分离菌的生化特性检验和特异性PCR鉴定表明，ZJ111／pcDNA3-SS能很好地侵入草鱼体内；对在Amp^ 、Amp^-平板上传5、10代和灌服7d后草鱼肝、脾脏分离的减毒菌抽提重组质粒进行PCR和酶切鉴定表明．减毒菌中的重组质粒在体外和侵入草鱼体内过程中具有较好的稳定性。     

蚓激酶重组逆转录病毒载体的构建及其在牛乳腺中的表达

将含有蚓激酶基因的表达盒插入到逆转录病毒载体pLNCL中获得了蚓激酶重组逆转录病毒载体,转染泌乳期奶牛乳腺小叶并获得暂时表达。利用脂质体转染PA317包装细胞,G418进行筛选得到了阳性细胞克隆,克隆扩大培养后病毒上清感染NIH3T3细胞进行滴度测定,最高滴度为1×104CFU/mL。进而用病毒上清转染妊娠期奶牛乳腺小叶组织,产后奶样经FAPA法检测表明蚓激酶基因已经整合到奶牛乳腺组织,并能实现相对稳定的表达。     

表达传染性喉气管炎病毒gB基因重组鸡痘病毒疫苗免疫持续期试验

将200003批疫苗免疫4周龄SPF鸡,免疫后的7、14、21、30、60、90、120、150、180和225 d分别采血,分离血清,采用ELISA方法检测血清抗FPV抗体,结果表明重组疫苗免疫后14 d,免疫鸡血清抗体已经全部阳转,免疫后的21 d血清抗FPV的抗体出现峰值,此后便开始下降,到免疫后的6个月抗体水平已经接近阴性对照的水平.用ILTV WG株和FPV 102株强毒进行的攻毒保护试验与血清检测的结果基本一致,在免疫后的5个月内可以使免疫鸡获得100%(10/10)保护,免疫后的6个月对ILT和FP的免疫保护分别为1/7和2/10,此时需要对鸡群实施二次免疫.其他5批疫苗(200001、200002、200101、200102和200103)免疫SPF鸡后5个月用ILTV WG株和FPV 102株攻击也获得了完全保护.     

Removal of Endotoxin from Swine Visfatin Recombinant Lipoprotein Solution

The purpose of this experiment was to explore the influence of the sample loading volume,flow rate and protein concentration of protein on the removal efficiency of endotoxin and protein loss in porcine visfatin recombinant lipoprotein solution at 4 ℃.In this study,commercial and modified polymyxin B was used as the medium to specifically remove endotoxin from the protein solution.At 4 ℃,different loading volume (6,8,10,12 mL),flow rate (0.125-0.75 mL/min) and protein concentration (0.9,1.2,1.3,1.7 mg/mL) were used for endotoxin removal,the content of endotoxin was determined by a quantitative method using endotoxin reagents,and the BCA protein concentration assay kit was used to determine the protein concentration.The results showed that at 4 ℃ endotoxin removal was the best when protein loading volume was 12 mL and flow rate was 0.125-0.25 mL/min,the removal rate was 99.5%,the residual amount of endotoxin (1 EU/mL) was within the biosafety range,and the protein recovery rate was as high as 90% to 95%.The experimental results showed that the endotoxin was successfully removed from the solution of porcine lipin recombinant protein,which provided references for the related functional research and clinical application of the protein.     

旋毛虫感染鼠血清P53抗体消长规律的观察

以纯化的旋毛形线虫P53排泄分泌重组蛋白包被微量反应板，建立检测旋毛虫抗体的间接ELISA方法，并以本方法测定旋毛形线虫感染的试验小鼠，试验显示，以100条肌幼虫/只经口饲喂感染25只昆明鼠，于第5天可检出抗体，第27天达到最高峰，第27天到第49天抗体水平维持稳定，随后抗体水平开始呈下降趋势。     

重组鹅β-防御素7蛋白的原核表达及其生物学特性分析

为探讨鹅β-防御素7(Av BD7)的生物学特性,将鹅Av BD7基因亚克隆到大肠杆菌原核表达载体p Pro EX HTa的EcoRⅠ和XhoⅠ双酶切位点上,构建重组表达质粒p Pro EX-Av BD7,将重组质粒转化到大肠杆菌Rosetta感受态细胞中,用异丙基-β-D-硫代半乳糖苷(IPTG)对其菌液进行诱导表达。经N-三(羟甲基)甲基甘氨酸十二烷基硫酸钠聚丙烯酰胺凝胶电泳(Tricine-SDS-PAGE)分析,该重组蛋白大小为10~15 ku,与预期大小结果一致。重组鹅Av BD7蛋白纯化后,通过菌落计数的方法测定其体外抗大肠杆菌、鸡白痢沙门氏菌、金黄色葡萄球菌、四联球菌、枯草芽孢杆菌等抑菌活性,盐离子浓度对其抗菌活性的影响及其对鸡红细胞的溶血活性。结果显示,重组鹅Av BD7蛋白对所测定的5种细菌均有显著抗菌活性(P0.05),且其抑菌活性随蛋白浓度的增加而增强。高浓度盐离子(150 mmol/L)显著抑制重组鹅Av BD7蛋白的抗菌活性(P0.05)。重组鹅Av BD7蛋白对鸡红细胞没有溶血活性(P0.05)。由此可见,重组鹅Av BD7蛋白具有广谱抗菌活性,高浓度盐离子显著降低其抗菌活性,且该重组蛋白不具有溶血活性。     

Construction and Analysis of Immune Response of Neospora caninum and Toxoplasma gondii Cross Recombinant Adenovirus Antigen AMA1 Gene

In order to establish AMA1 recombinant adenovirus of Neospora caninum (N.caninum) and Toxoplasma gondii (T.gondii), and analyze the immunogenicity of it, cross universal primers were designed according to the open reading frame of N. caninum and T. gondii AMA1 gene sequences. Based on pMD18T-NcAMA1 and pMD18T-TgAMA1 cloning plasmid, recombinant adenovirus shuttle plasmid ADV4-Nc/TgAMA1 was constructed. Then, ADV4-Nc/TgAMA1 and pacAd5 backbone plasmid were linearized and co-transfected 293T cells. After packaging recombinant adenovirus and measuring the virus titer, collected virus was inoculated into BALB/c mice, confirmed the IgG antibody levels by indirect ELISA method. The results showed that Nc/TgAMA1 was expressed in Ad5-Nc/TgAMA1 recombinant adenovirus, Ad5-Nc/TgAMA1 recombinant adenovirus titer was 10⁹ PFU/mL. IgG antibody levels in the Ad5-Nc/TgAMA1 vaccinated group were significantly higher than pVAX1-Nc/TgAMA1 plasmid group and PBS control group. This result indicated that the constructed Ad5-Nc/TgAMA1 recombinant adenovirus could induce specific humoral immune response in mice, this research laid a solid foundation for the development of a recombinant adenovirus vaccine against N. caninum and T. gondii.     

禽流感-新城疫重组二联活疫苗与重组禽流感灭活疫苗组合免疫效果观察

为进一步了解、掌握禽流感-新城疫重组二联活疫苗及与重组禽流感灭活疫苗组合使用的免疫效果,本研究以麻花鸡作为试验对象,制定了4种不同的免疫程序,分别对4组试验鸡群进行免疫,通过测定各组鸡只产生的免疫抗体效价和免疫抗体合格率,以评价二联活疫苗及与禽流感灭活疫苗的免疫效果。试验结果表明:禽流感-新城疫重组二联活疫苗单独用于肉鸡禽流感H5免疫预防抗体效价低于4 log2;建议禽流感-新城疫重组二联活疫苗可以与重组禽流感病毒灭活疫苗组合使用,两种疫苗免疫时间间隔2周左右,以免抑制机体产生对新城疫的免疫应答。     

PRRSV GP5和M蛋白重组腺病毒对猪的安全性和免疫效力

将猪繁殖与呼吸综合征病毒(PRRSV) GP5 重组腺病毒(recombinant Adenovirus-GP5,rAd-GP5)和M蛋白重组腺病毒(rAd‐M)分别接种4只30日龄PRRSV阴性断奶健康仔猪，结果为仔猪接种后2周内体温和食欲正常，猪血液、鼻塞、粪便和猪舍环境中重组腺病毒及其野生型腺病毒检测均为阴性。同时，将rAd‐GP5、rAd-M、rAd-GP5+M (rAd-GP5和rAd-M,V/V=1)和PRRSV-Resp弱毒株分别接种4只30日龄PRRSV阴性仔猪，不同时间检测PRRSV抗体，结果为免疫后14和45 d可检出PRRSV ELISA抗体和中和抗体；免疫后60～90 d，rAd-GP5和rAd-M免疫组ELISA抗体滴度均达1∶2400以上，与PRRSV‐Resp弱毒免疫组相似，而中和抗体滴度达1∶6以上，低于PRRSV-Resp弱毒免疫组；rAd-M免疫组ELISA抗体和中和抗体滴度则低于rAd‐GP5免疫组。rAd-GP5和rAd-M混合免疫组ELISA抗体达1∶4800,其中和抗体滴度与其单独免疫组相似。15只仔猪免疫后28 d进行攻毒保护试验，结果为重组腺病毒免疫猪攻毒后3 d平均体温为39.5℃,病毒血症和排毒时间仅为2周，与PRRSV-Resp弱毒免疫组相似，而空白对照组病毒血症和排毒时间至少60 d。攻毒后7～14 d，rAd-GP5和rAd‐M免疫组ELISA抗体和中和抗体水平明显升高。研究表明rAd-GP5和rAd-M重组腺病毒具有较好的安全性，均可诱导猪体产生较好的免疫反应，两者具有明显的协同免疫作用。     

猪α-干扰素临床促仔猪生长及疾病预防试验

选择广东四会某猪场40只断奶仔猪为观察对象,开展重组猪α-干扰素在促进生长作用和预防病毒性疾病方面的临床试验。试验1、2组与对照组相比较料重比分别降低6.3%和17.23%,平均每日增重率分别改变了-3.69%和24.85%,成活率分别提高了6.6%和14.3%,腹泻率分别降低了11.5%和26.9%,疾病总防治率分别提高了40.1%和55.49%;试验观察期间,对照组诊断为蓝耳病的4例,死亡1例,试验组则没有发生蓝耳病例。表明猪α-干扰素对仔猪的生长有良好的促进作用,具有显著的紧急预防病毒性疾病效果。