真核表达MERS-CoV刺突蛋白亚单位的信号肽序列优化研究
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摘要
中东呼吸综合征冠状病毒(Middle East respiratory syndrome coronavirus,MERS-CoV)的刺突蛋白(Spike,S)亚单位1(S1)是引起宿主免疫反应和产生中和抗体的主要靶抗原,也是疫苗研发和病原检测的重要靶标,选用适宜的真核表达系统高效表达S1蛋白是进行相关研究的基础。为确定MERS-CoV S1在哺乳动物细胞中高效分泌性表达的信号肽序列,构建了含高斯荧光素酶(Gaussia luciferase,GLuc)、人组织纤溶酶原激活剂(Tissue plasminogen activator,tPA)及小鼠免疫球蛋白G的2a亚型(Mouse immunoglobular G subtype 2a,MIgG2a)7个信号肽(原始序列和改造序列)序列的MERS-CoV S1表达质粒,瞬时转染细胞后,通过Western Blot检测并比较细胞培养上清和裂解液中S1的表达水平及分泌表达效率(条带密度灰度扫描比),并对哺乳动物细胞表达的S1蛋白的纯度与抗原特性进行了分析。结果表明7种信号肽在293T、BHK21和ExpiCHO-S~(TM)三种细胞系统中介导MERS-CoV S1的高效分泌表达的效率各有不同,其中tPA-1信号肽介导S1抗原在ExpiCHO-S~(TM)中具有较高的分泌表达效率与产量,纯化的S1蛋白保持了较好的抗原性。本研究为进一步研发基于MERS-CoV S1的亚单位疫苗及免疫学检测试剂奠定了基础。
MERS-CoV Spike(S) subunit 1(S1) is the main target antigen inducing immune response and neutralizing antibodies in the host, and it was preferred in vaccine development and serological detection. Efficient secretory expression of S1 is benificial for relevant researches. To optimize signal peptide sequences for efficient secretory expression of the MERS-CoV S1 in mammalian cell systems, seven DNA fragments were synthesized to encode native or modified versions of the signal peptide(SP) from Gaussian luciferase(GLuc), human tissue plasminogen activator(tPA) and mouse immunoglobular G subtype 2 a(MIgG2 a). SPs was fused to the5' end of the MERS-CoV S1 gene respectively to construct recombinant plasmids. And then the recombinant plasmids were transfected into cells, the expression and secretory efficiency of S1 in cell supernatant and lysates were analyzed by Western Blot and band grayscale ratio scanning. Furthermore, the purity and antigenic characteristic of S1 protein expressed in mammalian cells were analyzed by Western Blot and ELISA. The results showed that seven SPs promoted the secretory expression of MERS-CoV S1 in 293 T, BHK21 and ExpiCHOS~(TM) systems with different efficiency. MERS-CoV S1 mediated with tPA-1 SP showed higher level of secretory efficiency and yield in ExpiCHO-S~(TM). The purified MERS-CoV S1 maintained good antigenicity. This study lays the foundation for further development of subunit vaccines and immunological detection researches based on MERS-CoV S1 protein.
MERS-CoV Spike(S) subunit 1(S1) is the main target antigen inducing immune response and neutralizing antibodies in the host, and it was preferred in vaccine development and serological detection. Efficient secretory expression of S1 is benificial for relevant researches. To optimize signal peptide sequences for efficient secretory expression of the MERS-CoV S1 in mammalian cell systems, seven DNA fragments were synthesized to encode native or modified versions of the signal peptide(SP) from Gaussian luciferase(GLuc), human tissue plasminogen activator(tPA) and mouse immunoglobular G subtype 2 a(MIgG2 a). SPs was fused to the5' end of the MERS-CoV S1 gene respectively to construct recombinant plasmids. And then the recombinant plasmids were transfected into cells, the expression and secretory efficiency of S1 in cell supernatant and lysates were analyzed by Western Blot and band grayscale ratio scanning. Furthermore, the purity and antigenic characteristic of S1 protein expressed in mammalian cells were analyzed by Western Blot and ELISA. The results showed that seven SPs promoted the secretory expression of MERS-CoV S1 in 293 T, BHK21 and ExpiCHOS~(TM) systems with different efficiency. MERS-CoV S1 mediated with tPA-1 SP showed higher level of secretory efficiency and yield in ExpiCHO-S~(TM). The purified MERS-CoV S1 maintained good antigenicity. This study lays the foundation for further development of subunit vaccines and immunological detection researches based on MERS-CoV S1 protein.
引文
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[10] Futatsumori-Sugai M,Tsumoto K, Signal peptide design for improving recombinant protein secretion in the baculovirus expression vector system[J]. Biochem Biophys Res Commun, 2010, 391(1):931-935.
[11] Wen B, Deng Y, Guan J, Yan W, Wang Y, Tan W,Gao J. Signal peptide replacements enhance expression and secretion of hepatitis C virus envelope glycoproteins[J]. Acta Biochim Biophys Sin(Shanghai), 2011,43(2):96-102.
[12] Chen Y,Lu S,Jia H,Deng Y,Zhou J,Huang B,YuY, Lan J, Wang W, Lou Y, Qin K, Tan W. A novel neutralizing monoclonal antibody targeting the N-terminal domain of the MERS-CoV spike protein[J/OL].Emerg Microbes Infect, 2017, 6(5):e37.
[13] Jain N K, Barkowski-Clark S, Altman R, Johnson K,Sun F,Zmuda J,Liu C Y,Kita A,Schulz R,Neill A,Ballinger R,Patel R,Liu J,Mpanda A,Huta B,Chiou H, Voegtli W, Panavas T. A high density CHO-S transient transfection system:Comparison of ExpiCHO and Expi293[J]. Protein Expr Purif,2017, 134:38-46.
[2] World Health Organization. Middle East respiratory syndrome coronavirus(MERS-CoV)-Saudi Arabia[EB/OL]. http://www.who.int/csr/don/01-november-2018-mers-saudi-arabia/en/.
[3] Chen Y,Rajashankar K R,Yang Y,Agnihothram S S,Liu C, Lin Y L, Baric R S, Li F. Crystal structure of the receptor-binding domain from newly emerged Middle East respiratory syndrome coronavirus[J]. J Virol,2013, 87(19):10777-10783.
[4] Wirblich C, Coleman C M, Kurup D,Abraham T S,Bernbaum J G,Jahrling P B,Hensley L E,Johnson R F,Frieman M B, Schnell M J. One-health:a safe, efficient, dual-use vaccine for humans and animals against middle east respiratory syndrome coronavirus and rabies virus[J]. J Virol,2017, 91(2). pii:e02040-16.
[5] Chi H, Zheng X, Wang X, Wang C, Wang H, Gai W,Perlman S, Yang S, Zhao J, Xia X. DNA vaccine encoding Middle East respiratory syndrome coronavirus S1protein induces protective immune responses in mice[J].Vaccine, 2017, 35(16):2069-2075.
[6] Park W B,Perera R A,Choe P G,Lau E H,Choi S J,Chun J Y,Oh H S,Song K H,Bang J H,Kim E S,Kim H B, Park S W, Kim N J, Man Poon L L, Peiris M,Oh M D. Kinetics of serologic responses to MERS coronavirus infection in humans, South Korea[J].Emerg Infect Dis, 2015, 21(12):2186-2189.
[7] Wang W, Wang H, Deng Y, Song T, Lan J, Wu G,Ke C,Tan W. Characterization of anti-MERS-CoV antibodies against various recombinant structural antigens of MERS-CoV in an imported case in China[J/OL].Emerg Microbes Infect, 2016, 5(11):e113.
[8] Cheng G, Davis R E. An improved and secreted luciferase reporter for schistosomes[J]. Mol Biochem Parasitol,2007, 155(2):167-171.
[9] Wang J Y, Song W T, Li Y, Chen W J, Yang D,Zhong G C, Zhou H Z, Ren C Y, Yu H T, Ling H. Improved expression of secretory and trimeric proteins in mammalian cells via the introduction of a new trimer motif and a mutant of the tPA signal sequence[J]. Appl Microbiol Biotechnol, 2011, 91(3):731-740.
[10] Futatsumori-Sugai M,Tsumoto K, Signal peptide design for improving recombinant protein secretion in the baculovirus expression vector system[J]. Biochem Biophys Res Commun, 2010, 391(1):931-935.
[11] Wen B, Deng Y, Guan J, Yan W, Wang Y, Tan W,Gao J. Signal peptide replacements enhance expression and secretion of hepatitis C virus envelope glycoproteins[J]. Acta Biochim Biophys Sin(Shanghai), 2011,43(2):96-102.
[12] Chen Y,Lu S,Jia H,Deng Y,Zhou J,Huang B,YuY, Lan J, Wang W, Lou Y, Qin K, Tan W. A novel neutralizing monoclonal antibody targeting the N-terminal domain of the MERS-CoV spike protein[J/OL].Emerg Microbes Infect, 2017, 6(5):e37.
[13] Jain N K, Barkowski-Clark S, Altman R, Johnson K,Sun F,Zmuda J,Liu C Y,Kita A,Schulz R,Neill A,Ballinger R,Patel R,Liu J,Mpanda A,Huta B,Chiou H, Voegtli W, Panavas T. A high density CHO-S transient transfection system:Comparison of ExpiCHO and Expi293[J]. Protein Expr Purif,2017, 134:38-46.