【作者】 徐鹏；

【导师】 任林柱；

【作者基本信息】 吉林大学 ， 生物工程（专业学位）， 2022， 硕士

【摘要】 猪圆环病毒4型(Porcine circovirus type 4,PCV4)是一种新发现的无囊膜结构、单股环状正链的DNA病毒。自2019年以来,已经在韩国的部分城市和中国的17个省均检测出PCV4阳性样品。文献报道,可在各年龄段患有严重呼吸道疾病、腹泻和猪皮炎与肾病综合征等的病猪和健康猪中检测到PCV4。但是,PCV4的传染力、致病性、宿主范围等,及其对全球生猪养殖业造成的经济损失尚未知。因此,目前最迫切的就是研制出具有免疫原性,且安全有效的PCV4病毒疫苗,从而控制PCV4的发生和流行。PCV4的Cap蛋白是唯一的结构蛋白,含有多个抗原决定簇。因此,本实验选择Cap蛋白为靶蛋白,进行如下研究:(1)本研究利用原核表达的PCV4 Cap蛋白建立适用于临床与候选疫苗免疫原性检测的抗PCV4 Cap血清间接ELISA检测方法。本研究所建立的适用于临床与候选疫苗检测方法的临界值可分别达到0.262、0.157;特异性结果显示仅对PCV4血清呈现特异反应,对TGEV、PEDV、PDCo V、SVV、Ni V、PCV2、PCV3多种阳性血清无交叉反应;灵敏度可达1:102400。(2)根据PCV4的Cap蛋白特性设计适用于胞内表达的基因序列,分别进行相应表达系统密码子优化与基因合成。扩增密码子优化的Cap基因并将其连接到杆状病毒表达载体p Fast Bac1上,构建重组穿梭质粒,构建重组杆粒,转染贴壁SF9细胞获得具有感染力的重组杆状病毒r BV-PCV4,将其感染悬浮SF9细胞获得具有生物活性的PCV4 VLPs,构建具有良好免疫原性的PCV4 VLP疫苗;同时,将扩增的Cap基因连接到真核表达载体p VAX1上,转染293细胞中,经Western blot验证Cap蛋白成功表达,构建PCV4 DNA疫苗。将PCV4 VLP疫苗、DNA疫苗与相应对照组分别免疫小鼠后,进行免疫原性评价,检测体重、特异性抗体表达水平、T淋巴细胞分群状态、淋巴细胞增值情况、细胞因子表达水平。结果显示,PCV4 VLP疫苗、DNA疫苗可以使宿主产生明显针对PCV4的体液免疫反应和细胞免疫反应。PCV4 VLP疫苗主要通过分泌IFN-γ来刺激T淋巴细胞增殖分化,参与辅助细胞免疫。PCV4 DNA疫苗主要通过分泌IL-2来刺激CD8~+T淋巴细胞分化为CTL细胞,引起并增强细胞免疫反应。综上所述,本研究基于PCV4 Cap蛋白成功构建了PCV4 VLP疫苗和DNA疫苗,并建立了特异性抗体检测方法。免疫实验显示,两种疫苗均可以有效引起体液免疫与细胞免疫应答。本研究为深入开展PCV4临床诊断、新型疫苗研发奠定了基础,提供新思路。更多还原

【Abstract】 Porcine circovirus type 4(PCV4)is a newly discovered DNA virus with no envelope structure and single strand circular positive strand.Since 2019,PCV4 positive samples have been detected in some cities in South Korea and 17 provinces in China.It is reported that PCV4 can be detected in healthy pigs and sick pigs with severe respiratory diseases,diarrhea,porcine dermatitis and nephrotic syndrome at all ages.However,the infectivity,pathogenicity and host range of PCV4 and its economic losses to the global pig breeding industry are unknown.Therefore,at present,the most urgent thing is to develop a safe and effective PCV4 virus vaccine with immune efficacy,so as to control the occurrence and prevalence of PCV4.Cap protein encoded by PCV4 is the only structural protein containing multiple antigenic determinants.Therefore,in this experiment,Cap protein was selected as the target protein for the following research:(1)This study also used the prokaryotic expression of PCV4 Cap protein to establish an indirect ELISA method for the detection of anti PCV4 Cap serum,which is suitable for the immunogenicity of clinical and candidate vaccines.The critical values for clinical and candidate vaccine detection methods established in this study can reach 0.262 and 0.157 respectively;The specificity results showed that only PCV4 showed specific reaction,and there was no cross reaction to TGEV,PEDV,PDCo V,SVV,Ni V,PCV2 and PCV3;The sensitivity can reach 1:102400.(2)According to the sequence characteristics of PCV4 Cap gene,the gene sequence suitable for intracellular expression is designed,the codon of the corresponding expression system is optimized.The codon optimized cap gene is amplified and connected to the baculovirus expression vector,p Fast Bac1 and to construct the recombinant shuttle plasmid and the recombinant rod to obtain the infectious recombinant baculovirus r BV-PCV4.After infecting Sf9adherent cells,PCV4 Cap VLP with biological activity was successfully obtained to construct PCV4 VLP vaccine with immune efficacy;The amplified and optimized Cap gene was connected to eukaryotic expression vector that named as p VAX1 and transfected into 293 cells.Western blot verified that Cap protein was successfully expressed.Consequently,p VAX1-PCV4 was constructed PCV4 DNA vaccine.After the mice were immunized with PCV4 VLP vaccine,DNA vaccine and corresponding control groups,the body weight,the expression level of specific antibody,the clustering status of CD4~+T lymphocytes,CD8~+T lymphocytes,the proliferation of lymphocytes and the expression level of cytokines were detected.The results showed that PCV4VLP vaccine and DNA vaccine could make the host produce obvious humoral immune response and cellular immunity response against PCV4.PCV4 VLP vaccine is mainly secreted by IFN-γto stimulate the proliferation and differentiation of T lymphocytes to participate in and assist cellular immunity.PCV4 DNA vaccine mainly secreted IL-2 to stimulate CD8~+T lymphocytes to differentiate into CTL cells,causing and enhancing cellular immune response.In conclusion,in this study,PCV4 VLP vaccine and DNA vaccine were successfully constructed based on PCV4 Cap protein,and a specific antibody detection method was established.Immune experiments show that both vaccines can effectively induce humoral and cellular immune responses.This study has laid a foundation and provided new ideas for the further development of pcv4 clinical diagnosis and new vaccine research and development.更多还原