为构建以新城疫病毒（NDV）为活毒载体表达鸡传染性支气管炎病毒（IBV）S基因的重组病毒,本研究利用RT-PCR技术,以IBV的Massachusetts41株RNA为模板,通过RT-PCR扩增得到IBV S基因（3534bp）,将其插入到NDV感染性克隆pBRN-FL中,构建了含有IBV S基因的重组NDV cDNA克隆pBRN-FL-IBVS。利用磷酸钙转染法,在辅助质粒pBS-NP、pBS-P和pBS-L的共同作用下,将pBRN-FL-IBVS转染表达T7聚合酶重组痘病毒感染的BSR细胞,救获重组NDV（rL-IBVS）。采用RT-PCR检测接种重组病毒的鸡胚尿囊液,结果表明rL-IBVS中含有相应外源基因。IFA试验表明,rL-IBVS可与鸡抗IBV的高免血清发生特异性反应,证明S蛋白在感染的BSR细胞中得到表达。其鸡胚平均致死时间、脑内致病指数和静脉内致病指数等指标显示rL-IBVS保持了亲本疫苗株高滴度的鸡胚生长特性和低致病力特性。本研究采用反向遗传操作技术构建了表达IBV S蛋白的重组NDV,为进一步研制IBV和NDV的重组基因工程活载体疫苗奠定了基础。

Newcastle disease virus (NDV) is an ideal model for exploring the mechanisms of the virus; it is also an optimal vector for developing vector vaccines and for cancer therapy. A reverse genetic system of NDV Mukteswar strain controlled by eukaryotic cellular RNA polymerase II promoter was established by reverse genetics technology. Based on the reverse genetic system, an open reading frame of the enhanced green fluorescent protein (EGFP) gene be inserted between the P and M genes of the viral genome and flanked with the gene start (GS) sequence and gene end (GE) sequence to form an independent transcription unit. The rescued virus was amplified in specific pathogen-free (SPF) chicken embryos for 10 generations, and the results showed that the recombinant virus could stably express the exogenous gene for at least 10 generations. Efficient expression of two exogenous genes synchronously is essential for the development of NDV-based multivalent vaccine candidates. Explore the possibility of simultaneous and efficient expression of two exogenous genes based on NDV vector. In the present study, a recombinant virus with co-expression of EGFP and cherry fluorescent protein (CFP) inserted between the intergenic regions of the P/M gene as two independent transcription units was successfully rescued. The results showed that the two exogenous genes could be expressed synchronously and efficiently. The results of biological analysis of the expression efficiency of exogenous genes showed that the EGFP in recombinant viruses with two exogenous genes was slightly lower than that of recombinant viruses with one exogenous gene, but the expression efficiency of CFP in recombinant viruses with two exogenous genes was higher than EGFP in both viruses. These recombinant viruses have similar growth kinetics but with a little attenuation in virulence compared with parental viruses. In conclusion, these data indicated that this study successfully established the reverse genetic system of the NDV Mukteswar strain and achieved the purpose of efficient expression of two exogenous genes synchronously in a novel approach, laying the foundation for the development of multivalent vaccines or tumor therapeutics using NDV as a vector.

为构建表达猪圆环病毒2型（PCV2）衣壳蛋白（Cap）的重组新城疫病毒（NDV）,本研究利用NDV La Sota弱毒疫苗株为活病毒载体,通过反向遗传操作系统构建出表达PCV2 Cap的重组病毒（r La-PCV2/Cap）,以及表达删除核定位信号（NLS）的Cap（CapΔ41）的重组病毒（r La-PCV2/CapΔ41）,并对外源蛋白的表达效果进行了检测。通过间接免疫荧光试验证明,两种重组病毒感染的BHK-21细胞中,PCV2 Cap蛋白以及删除NLS的CapΔ41蛋白均获得正确表达;其中,完整的Cap蛋白定位于核内,而删除NLS的CapΔ41蛋白则定位于胞浆中,本研究结果为研究新型PCV2疫苗奠定基础。

为构建表达H5N1亚型禽流感病毒（AIV）HA蛋白重组新城疫病毒（NDV）及比较密码子优化的HA免疫增强作用,本研究利用NDV LaSota弱毒疫苗株的反向遗传操作系统,分别构建了表达野毒型H5N1亚型AIV HA蛋白和密码子优化的HA蛋白的重组NDV,并分别命名为rL-H5HA和rL-optiH5HA。对拯救的重组病毒进行RT-PCR、间接免疫荧光和western blot鉴定,结果表明,两种重组病毒均可以在BHK-21细胞中以及鸡胚成纤维细胞稳定地表达HA蛋白。此外,rL-optiH5HA在体外增殖能力及在体内刺激产生抗体均显著高于rL-H5HA。本研究表明,密码子优化的HA重组NDV具有明显的免疫增强作用。本研究为研制安全、有效的新型禽流感疫苗提供了实验依据。

Development of a safe and efficient in-ovo vaccine against Newcastle disease (NDV) and very virulent infectious bursal disease virus (vvIBDV) is of great importance. In this study, a chimeric NDV LaSota virus with the L gene of Clone-30 (rLaC30L) was used to generate a recombinant chimeric virus expressing the VP2 protein of vvIBDV (rLaC30L-VP2). The safety and efficacy of rLaC30L-VP2 in-ovo vaccination was then evaluated in 18-day-old special pathogen free (SPF) chicken embryos and commercial broiler embryos for prevention of NDV and vvIBDV. Hatchability and global survival rate of the hatched birds was not affected by in-ovo rLaC30L-VP2 vaccination. However, rLaC30L-VP2 in-ovo vaccination induced significant anti-IBDV and anti-NDV antibodies in SPF birds and commercial broilers, and 100% of vaccinated chickens were protected against a lethal NDV challenge. In-ovo rLaC30L-VP2 vaccination also provided resistance against vvIBDV challenge in a significant amount of animals. These results suggest that rLaC30L-VP2 is a safe and efficient bivalent live in-ovo vaccine against NDV and vvIBDV. (C) 2014 Elsevier Ltd. All rights reserved.

为研制安全、有效的新型鸡毒支原体（MG）疫苗,本研究利用新城疫病毒（NDV）LaSota弱毒疫苗株反向遗传操作系统,构建出表达MG TM1蛋白的重组病毒rLa-TM1,并对其生物学特性进行鉴定,评估其作为MG活载体疫苗的安全性和有效性。通过免疫荧光试验表明TM1蛋白获得正确表达;经测定,重组病毒平均鸡胚致死时间（MDT）为132 h,脑内致死指数（ICPI）和静脉内致病指数（IVPI）均为0,保持了LaSota亲本株的低致病性;rLa-TM1接种鸡胚所收集尿囊液中的病毒滴度可高达每毫升109.5EID50以上,证明重组病毒保持了LaSota亲本株高滴度鸡胚生长特性;重组病毒rLa-TM1接种1周龄的雏鸡后,可以诱导显著的MG抗体反应;攻毒保护试验证明其保护率可以达90%,而对照组全部发病（10/10）。本研究表明重组病毒rLa-TM1可以作为MG重组病毒活载体疫苗的候选疫苗。

为研制安全、有效的新型犬瘟热疫苗,本研究利用新城疫病毒（NDV）LaSota弱毒疫苗株反向遗传操作系统,构建出表达犬瘟热病毒（CDV）弱毒疫苗株Rockborn-20/8血凝素（H）蛋白的重组病毒rLa-CDV-H,并对其生物学特性进行鉴定,评估其作为犬瘟热活载体疫苗的安全性和有效性。通过免疫荧光和western blot试验证明了CDV H蛋白的正确表达;重组病毒株保持了LaSota亲本株的低致病性和高滴度鸡胚生长特性;重组病毒rLa-CDV-H接种12周龄比格犬后,可以诱导显著的CDV中和抗体反应。本研究表明重组病毒rLa-CDV-H具有作为犬瘟热重组病毒活载体疫苗的潜力。

为获得鸡传染性支气管炎病毒（IBV）M41株的全长S1蛋白,并以S1蛋白为包被抗原建立间接ELISA抗体检测方法,试验采用RT-PCR方法扩增出S1基因,将其克隆到pBluescriptⅡKS（+）载体中,酶切、测序鉴定后将S1基因导入融合表达载体pMAL-c2X中,将重组质粒转化大肠杆菌TB1,IPTG诱导后,应用SDS-PAGE及Western-blot检测蛋白表达情况。结果表明:融合蛋白得到了表达;以纯化的重组蛋白作为包被抗原,在包被量为3.2μg/孔、血清160倍稀释条件下P/N值最大,初步建立了IBV抗体间接ELISA检测方法。