猪圆环病毒3型Cap蛋白多肽合成及免疫原性分析  

Peptide synthesis and immunogenicity analysis of Porcine circovirus type 3 Cap protein

作　　者：贺会利 冯世文[1] 李军[1] 胡帅[1] 禤雄标[1] 谢永平[1] 柳锋[1] 钟舒红[1] 庞彬辉[1] 潘艳[1,2] HE Huili;FENG Shiwen;LI Jun;HU Shuai;XUAN Xiongbiao;XIE Yongping;LIU Feng;ZHONG Shuhong;PANG Binhui;PAN Yan(Guangxi Veterinary Research Institute,Nanning 530001,China;Guangxi Vocational Technical College,Nanning 530001,China)

机构地区：[1]广西兽医研究所,南宁530001 [2]广西农业职业技术学院,南宁530001

出　　处：《黑龙江畜牧兽医》2021年第23期78-82,87,154,共7页Heilongjiang Animal Science And veterinary Medicine

基　　金：广西科技重大专项(桂科AA17204057);广西科技基地和人才专项(桂科AD17195015);广西兽医生物技术重点实验室自主研究课题项目(17-259-36-A-2)。

摘　　要：为了筛选出具有较强免疫原性的猪圆环病毒3型(Porcine circovirus virus type 3,PCV-3)核衣壳(Cap)蛋白多肽,试验应用生物信息学技术,通过Garnier-Robson和Chou-Fasman两种方法分析PCV-3-Cap蛋白的二级结构,Kyte-Doolittle方法分析亲水性,Karplus-Schulz方法分析柔性,Emini方法分析表面可能性,Jameson-Wolf方法分析抗原性,经对该蛋白生物学特性进行综合分析设计合成PCV-3-Cap蛋白多肽,与钥孔血蓝蛋白(KLH)偶联后作为抗原免疫小鼠,测定免疫后小鼠抗体效价以及血清中免疫球蛋白IgG、IgA和细胞因子干扰素-γ(IFN-γ)、白细胞介素-6(IL-6)、白细胞介素-4(IL-4)的含量。结果表明:PCV-3-Cap蛋白的α-螺旋位于1~29,59~72,77~83,133~142,181~189 aa等区域;β-折叠位于1~15,20~33,37~52,62~111,114~123,131~155,161~167,175~193,203~215 aa等区域;转角位于12~15,32~43,137~144,167~170,174~177 aa等区域;无规则卷曲位于10~12,53~75,113~130,156~159,169~178,202~204 aa等区域;0~46,115~145 aa区域的亲水指数较高;柔性结构主要位于9~21,122~133,152~162 aa等区域;7~30,32~45,54~62,119~148 aa这些区域暴露于蛋白质表面的可能性较大;6~46,121~151 aa区域的抗原指数较高,经综合分析筛选出2段抗原性较好的肽段,分别为30~45 aa(16K)和130~145 aa(17K);2条PCV-3-Cap蛋白多肽均在第4次免疫小鼠后抗体效价达到1∶6 400;四免后小鼠的免疫球蛋白IgG、IgA及细胞因子IFN-γ、IL-6、IL-4含量与对照组相比均显著提高(P<0.05)。说明筛选出的2个PCV-3-Cap蛋白多肽具有较好的抗原性。In order to screen out Porcine circovirus virus type 3(PCV-3) nucleocapsid(Cap) protein peptides with strong immunogenicity, the experiment used bioinformatics technology to analyze the secondary structure of PCV-3-Cap protein by Garnier-Robson and Chou-Fasman method, to analyze its hydrophilicity by using Kyte-Doolittle method, to analyze flexible zone by using Karplus-Schulz method, to analyze the surface possibility by using the Emini method, and to analyze antigenicity by using the Jameson-Wolf method. After comprehensive analysis, design and synthesis of PCV-3-Cap protein peptides were carried out, which were coupled with keyhole limpet hemocyanin(KLH) as an antigen to immunize mice. The antibody titers of mice after immunization and the serum levels of immunoglobulin IgG, IgA, interferon-γ(IFN-γ), interleukin-6(IL-6), and interleukin-4(IL-4) contents were determined. The results showed that the α-helix of PCV-3-Cap protein was located at 1-29, 59-72, 77-83, 133-142, 181-189 aa and other zones;β-sheet was located at 1-15, 20-33, 37-52, 62-111, 114-123, 131-155, 161-167, 175-193, 203-215 aa and other zones;the corner was located at 12-15, 32-43, 137-144, 167-170, 174-177 aa and other zones;random coil was located at 10-12, 53-75, 113-130, 156-159, 169-178, 202-204 aa and other zones. The 0-46, 115-145 aa zone had a higher hydropathic index;the flexible structure was mainly located in 9-21, 122-133, 152-162 aa and other zones;these zones of 7-30, 32-45, 54-62, 119-148 aa were more likely to be exposed to the protein surface;the zone of 6-46, 121-151 aa had a higher antigen index. After comprehensive analysis, two peptides with good antigenicity were selected, which were 30-45 aa(16 K) and 130-145 aa(17 K), respectively. Both PCV-3-Cap protein peptides had the highest antibody titer after the fourth immunization of mice, reaching 1∶6 400;after the fourth immunization, the contents of immunoglobulin IgG, IgA and cytokines IFN-γ, IL-6 and IL-4 in mice were significantly increased compared with those in the

关 键 词：猪圆环病毒3型 CAP蛋白 多肽 抗体 免疫原性 

分 类 号：S852.659.2[农业科学—基础兽医学] S828[农业科学—兽医学]