甘露糖修饰的壳聚糖聚乳酸-羟基乙酸共聚物纳米微球作为口蹄疫病毒核酸疫苗递送载体的特性评价

旨在对制备的甘露糖修饰的壳聚糖聚乳酸-羟基乙酸共聚物[poly （D，L-lactide-co-glycolide），PLGA]纳米微球作为口蹄疫病毒（foot-and-mouth disease virus，FMDV）核酸疫苗递送载体进行评价。采用西佛碱反应和元素分析制备具有一定取代度的甘露糖修饰的壳聚糖衍生物（mannose modified chitosan，MCS），然后，经双重乳化挥发法制备得到甘露糖修饰的壳聚糖PLGA纳米微球（MCS-PLGA-NPs）。采用纳米粒径仪检测MCS-PLGA-NPs粒径分布和表面电势（zeta）、扫描电镜考察其形态、琼脂糖凝胶电泳观察其对质粒的吸附和吸附质粒后抵抗核酸酶降解能力、CCK-8法检测MCS-PLGA-NPs的细胞毒性、激光共聚焦观察巨噬细胞对MCS-PLGA-NPs-质粒DNA复合物的摄取、荧光显微镜和Western blot验证MCS-PLGA-NPs加载质粒DNA在细胞中的表达。元素分析结果表明，成功制备了取代度为5%~10%的MCS。纳米粒径测定和扫描电镜结果表明，MCS-PLGA-NPs的zeta为正值、粒径分布均匀且形态规则呈球形。琼脂糖凝胶电泳结果显示，MCS-PLGA-NPs吸附质粒的能力随着其质量的增加而增强并且可以在一定程度上抵抗核酸酶降解质粒DNA。在细胞毒性试验中，不同浓度的MCS-PLGA-NPs与RAW264.7细胞共孵育24 h后，细胞存活率仍在85%以上。在细胞摄取试验中，用激光共聚焦显微镜可以明显观察到质粒DNA结合到纳米微球表面被RAW264.7细胞摄取。荧光显微镜和Western blot试验证明MCS-PLGA-NPs加载质粒DNA可以在细胞中进行表达。综上表明，本研究成功制备了MCS以及具有递送核酸疫苗能力的MCS-PLGA-NPs，为FMDV核酸疫苗的递送研究提供了新的方向和见解，也为该递送载体携带特定抗原靶向抗原递呈细胞表面甘露糖受体以及应用于动物免疫的研究奠定基础。     

Protective efficacy of an H5/H7 trivalent inactivated vaccine produced from Re-11, Re-12, and H7-Re2 strains against challenge with different H5 and H7 viruses in chickens

We developed an H5/H7 trivalent inactivated vaccine by using Re-11, Re-12, and H7-Re2 vaccine seed viruses, which were generated by reverse genetics and derived their HA genes from A/duck/Guizhou/S4184/2017(H5 N6)(DK/GZ/S4184/17)(a clade 2.3.4.4 d virus), A/chicken/Liaoning/SD007/2017(H5 N1)(CK/LN/SD007/17)(a clade 2.3.2.1 d virus), and A/chicken/Guangxi/SD098/2017(H7 N9)(CK/GX/SD098/17), respectively. The protective efficacy of this novel vaccine and that of the recently used H5/H7 bivalent inactivated vaccine against different H5 and H7 N9 viruses was evaluated in chickens. We found that the H5/H7 bivalent vaccine provided solid protection against the H7 N9 virus CK/GX/SD098/17, but only 50–60% protection against different H5 viruses. In contrast, the novel H5/H7 trivalent vaccine provided complete protection against the H5 and H7 viruses tested. Our study underscores the importance of timely updating of vaccines for avian influenza control.     

口蹄疫疫苗对牛的应用效果调查

牛口蹄疫是传染性疾病,为研究青海省峰堆乡口蹄疫疫苗的免疫效果,于2018—2019年对该乡的7个村开展牛口蹄疫疫苗免疫效果调查,共调查牛634头,7个村总体的口蹄疫合格率为100.00%和97.56%,最低合格率为81.63%和88.63%。为更好地控制该类疾病发生流行,提升免疫效果,该文探讨口蹄疫疫苗免疫失败原因,并制定了相应的预防措施。     

Construction of recombinant capripoxviruses as vaccine vectors for delivering foreign antigens: Methodology and application

Goatpox (GTP), sheeppox (SPP) and lumpy skin disease (LSD) are three severe diseases of goat, sheep and cattle. Their typical clinical symptoms are characterized by vesicles, papules, nodules, pustules and scabs on animal skins. The GTP, SPP and LSD are caused by goatpox virus (GTPV), sheeppox virus (SPPV) and lumpy skin disease virus (LSDV), respectively, all of which belong to the genus Capripoxvirus in the family Poxviridae. Several capripoxvirus (CaPV) isolates have been virulently attenuated through serial passaging in vitro for production of live vaccines. CaPV-based vector systems have been broadly used to construct recombinant vaccines for delivering foreign antigens, many of which have been demonstrated to induce effective immune protections. Homologous recombination is the most commonly used method for constructing recombinant CaPVs. Here, we described a methodology for generation of recombinant CaPVs by the homologous recombination, and further reviewed CaPV-vectored vaccines for delivering foreign antigens.     

牛支原体新疆分离株P48基因的克隆、原核表达及重组蛋白免疫原性研究

试验旨在确定牛支原体P48基因的免疫原性，为进一步筛选牛支原体免疫保护性基因奠定基础。本研究以牛支原体新疆分离株为研究对象，运用Overlap PCR方法扩增得到点突变后的牛支原体新疆分离株P48基因，构建原核表达载体pET-32a （+）-P48，转化大肠杆菌BL21（DE3）感受态细胞，在诱导剂ITPG的诱导下获得重组蛋白P48，纯化后的重组P48蛋白免疫BALB/c小鼠制备多克隆抗体，运用Western blotting和ELISA方法验证其反应原性和免疫原性。结果表明，试验成功构建原核表达载体pET-32a （+）-P48，重组蛋白P48大小约为66 ku，纯化后的牛支原体P48重组蛋白免疫小鼠后可产生良好的免疫反应，血清抗体滴度达到较高水平（D_{450 nm}值为1.126）。Western blotting结果显示，抗牛支原体P48重组蛋白的鼠血清与牛支原体P48重组蛋白及牛支原体全菌蛋白抗原均能产生明显的抗原抗体反应，表明P48重组蛋白具有良好的免疫原性与反应原性，可作为牛支原体新型疫苗的候选基因，且牛支原体新疆分离株P48基因与国内外5株牛支原体P48基因的同源性很高，亲缘关系较近。     

A simplified roller bottle platform for the production of a new generation VLPs rabies vaccine for veterinary applications

Rabies is a neglected disease with an estimated annual mortality of 55,000 human deaths, affecting mainly low-income countries. Over 95% of these cases result from virus transmission through the bite of infected dogs and for this reason there is a real need for a cheap and effective rabies veterinary vaccine to be used in mass vaccination campaigns. In this work, we describe the establishment of a simple platform for the production of a virus-like particles based rabies vaccine using mammalian cells and roller bottles as culture system. Adherent cells were cultured during more than 15 days and VLPs were continuously produced and secreted to the culture supernatant. Immunogenicity and protective efficacy of VLPs were tested through rabies virus neutralizing antibody test and NIH potency test. These viral particles induced high titer of long lasting neutralizing antibodies and protected mice against active virus challenge. Therefore, this development represents a promising platform for the production of a new generation and virus-free rabies vaccine candidate for veterinary applications.     

川西地区鸡滑液囊支原体分离株的部分生物学特性及其VlhA基因遗传进化分析

为了解鸡滑液囊支原体(MS)在川西地区的感染情况,本研究采集15个肉鸡场疑似MS感染的病鸡咽拭子、跗关节和胸部滑液囊样本共75份,对经PCR检测为阳性的样本进行MS分离,并对分离株的主要生物学特性进行研究,以及对分离株的VlhA基因进行遗传进化分析。结果显示,75份样本MS PCR阳性检出率为41.33%(31/75),11个鸡场感染该病,场阳性率为73.33%(11/15);但仅从其中3个鸡场分离到9株MS,分离株菌落与菌体形态与已知MS培养特性相符,各分离株培养浓度为10~4CCU/mL^10~6CCU/mL,分离株以5×10~5CCU接种7日龄SPF鸡胚,鸡胚于接种后7 d^9 d死亡,且分离回收到接种菌株;9株MS分离株VlhA基因的同源性为86.1%~99.9%,与参考株同源性为86.2%~95.4%,其中分离自同一鸡场的7株MS VlhA基因同源性为96.4%~99.9%;分离株VlhA基因遗传进化分析显示,其中分离自两个不同鸡场的两株MS与国内流行株的亲缘关系最近,而分离自另一鸡场的7株MS与中东地区分离的3株MS亲缘关系较近,表明MS在川西地区肉鸡场呈高感染率,MS VlhA基因变异较大。本研究为川西地区MS的进一步研究提供了基础材料和科学依据。