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小鼠PD-1及其配体PD-L1胞外区基因的原核表达及亲和性

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温州医科大学学报 2018年第6期48卷 401-407,412页

作者：吕开绩陈旭东程开王路得朱进顺 kamara Saidu 张丽芳朱冠保温州医科大学附属第一医院胃肠外科浙江温州325015 温州医科大学分子病毒与免疫研究所微生物学与免疫学教研室浙江温州325035

目的:制备小鼠PD-1胞外区(mPD-1)与其配体PD-L1胞外区(mPD-L1)原核表达蛋白,并制备mPDL1兔多克隆抗体,在体外对mPD-L1和mPD-1的结合亲和性进行验证。方法:通过分子克隆方法分别构建重组质粒pET21a/mPD-L1和p GEX4T-1/mPD-1,并通过原核... 详细信息

目的:制备小鼠PD-1胞外区(mPD-1)与其配体PD-L1胞外区(mPD-L1)原核表达蛋白,并制备mPDL1兔多克隆抗体,在体外对mPD-L1和mPD-1的结合亲和性进行验证。方法:通过分子克隆方法分别构建重组质粒pET21a/mPD-L1和p GEX4T-1/mPD-1,并通过原核表达系统制备相应的重组蛋白并纯化,通过Western blot进行鉴定;将纯化的mPD-L1蛋白免疫健康日本大耳白兔制备兔多克隆血清抗体,用ELISA、Western blot分析得到的多克隆抗体的效价和特异性,并且将该多克隆抗体作为免疫荧光的阳性对照。最后应用ELISA和免疫荧光验证mPD-1与其配体mPD-L1蛋白的亲和性。结果:SDS-PAGE和Western blot结果显示原核表达的重组蛋白与预期大小一致,mPD-L1和mPD-1分别为25 kDa和40 kDa。经mPD-L1蛋白免疫后的日本大耳白兔能够产生特异性抗体,抗体效价在免疫后第6周达到高峰,Western blot检测结果显示多克隆血清可特异性识别mPD-1。ELISA结果表明,原核表达的mPD-L1蛋白能够和mPD-1蛋白结合,实验组OD450值明显高于对照组。免疫荧光结果显示,在小鼠PD-L1阳性细胞B16(小鼠黑色素瘤细胞株)的胞膜区出现绿色荧光团块。结论:利用原核细胞表达并纯化后获得mPD-L1和mPD-1蛋白,并在体外成功验证了mPD-L1和mPD-1之间的结合特性,为后期利用mPD-1和PD-L1的生物学特性研究提供了基础。

关键词： mPD-1 mPD-L1 原核表达干扰素 ELISA 免疫荧光小鼠

新型冠状病毒E蛋白结构与免疫优势表位的信息学预测分析

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温州医科大学学报 2021年第3期51卷 209-214,219页

作者：李明洋 SAIDU kamara 汪琪郭艳茹李青峰朱珊丽陈俊蒋朋飞张丽芳温州医科大学分子病毒与免疫研究所病原生物与免疫学系浙江温州325035

目的:应用生物信息学方法预测和分析新型冠状病毒(SARS-CoV-2)包膜蛋白(E蛋白)的结构及免疫优势表位。方法:从NCBI GenBank数据库中检索SARS-CoV-2 E蛋白的氨基酸序列,通过Expas y服务器上的软件分析预测其理化性质、二级结构、跨膜区域... 详细信息

目的:应用生物信息学方法预测和分析新型冠状病毒(SARS-CoV-2)包膜蛋白(E蛋白)的结构及免疫优势表位。方法:从NCBI GenBank数据库中检索SARS-CoV-2 E蛋白的氨基酸序列,通过Expas y服务器上的软件分析预测其理化性质、二级结构、跨膜区域;并结合其亲水性、表面可及性、极性、抗原性和柔韧性等综合分析,预测其可能的B细胞表位;利用Net CTL1.2 server预测CTL表位;通过Vector NTI对冠状病毒属E蛋白进行同源性分析;同时应用Swiss Model对E蛋白进行三维结构同源建模及功能结构域预测分析。结果:SARS-CoV-2 E蛋白由75个氨基酸残基组成,为跨膜蛋白,跨膜区域以α螺旋为主;E蛋白可能含有2个B细胞表位和6个CTL表位,其免疫优势表位区域分别位于其N端16-34aa和C端50-70aa区段;SARS-CoV-2与SARS-CoV和Bat-SARS-like-CoV E蛋白的免疫优势表位存在极高的同源性;其疏水性跨膜结构和PDZ结合基序分别位于N端12-34aa和C端72-75aa区段。结论:E蛋白为跨膜蛋白,其氨基酸序列的16-34和50-70区段可能为免疫优势表位。

关键词：新型冠状病毒包膜蛋白生物信息学免疫优势表位同源性分析

Intra-host Ebola viral adaption during human infection

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Biosafety and Health 2019年第1期1卷 14-24页

作者： William JLiu Weifeng Shi Wuyang Zhu Cong Jin Shumei Zou Ji Wang Yuehua Ke Xiaofeng Li Mi Liu Tao Hu Hang Fan Yigang Tong Xiang Zhao Wenbin Chen Yuhui Zhao Di Liu Gary Wong Chengchao Chen Chunyu Geng Weiwei Xie Hui Jiang Idrissa Laybor kamara Abdul kamara Matt Lebby Brima Kargbo Xiangguo Qiu Yu Wang Xiaofeng Liang Mifang Liang Xiaoping Dong Guizhen Wu George F.Gao Yuelong Shu NHC Key Laboratory of Biosafety National Institute for Viral Disease Control and PreventionChinese Center for Disease Control and PreventionBeijing 102206China Shandong Universities Key Laboratory of Etiology and Epidemiology of Emerging Infectious Diseases Taishan Medical UniversityTaian 271000China Institute of Disease Control and Prevention Academy of Military Medical SciencesBeijing 100071China State Key Laboratory of Pathogen and Biosecurity Institute of Microbiology and EpidemiologyAcademy of Military Medical SciencesBeijing 100071China CAS Key Laboratory of Pathogenic Microbiology and Immunology Institute of MicrobiologyChinese Academy of SciencesBeijing 100101China BGI-Shenzhen Shenzhen 518083China Sierra Leone Ministry of Health and Sanitation FreetownSierra Leone Special Pathogens Program National Microbiology LaboratoryPublic Health Agency of CanadaWinnipegCanada Chinese Center for Disease Control and Prevention Beijing 102206China Public Health School(Shenzhen) Sun Yat-sen UniversityShenzhen 510006China

The onsite next generation sequencing(NGS)of Ebola virus(EBOV)genomes during the 2013–2016 Ebola epidemic in Western Africa provides an opportunity to trace the origin,transmission,and evolution of this ***,we have diagnosed a cohort of EBOV patients in Sierra Leone in 2015,during the late phase of the *** surviving EBOV patients had a recovery process characterized by decreasing viremia,fever,and biochemical *** genomes sequenced through the longitudinal blood samples of these patients showed dynamic intra-host substitutions of the virus during acute infection,including the previously described short stretches of 13 serial TNC ***,within individual patients,samples collected during the early phase of infection possessed Ts at these nucleotide sites,whereas they were replaced by Cs in samples collected in the later phase,suggesting that these short stretches of TNC mutations could emerge *** addition,up to a total of 35 nucleotide sites spanning the EBOV genome were mutated *** study showed the dynamic intra-host adaptation of EBOV during patient recovery and gave more insight into the complex EBOV-host interactions.

关键词： Ebola virus Intra-host Nucleotide Variation Adaptation Genome sequencing Clinical manifestations Blood biochemistry