Detection of Interaction Between Porcine Type I Complement Receptor and C3b Active Fragment in Vitro
SUN YuChen,, JIA RuiPu, FAN KuoHai, SUN Na, SUN YaoGui, SUN PanPan, LI HongQuan,, YIN Wei,College of Veterinary Medicine, Shanxi Agricultural University, Taigu 030801, Shanxi
Abstract 【Objective】In order to provide scientific data for elucidating the molecular mechanism of porcine erythrocyte immune adhesion function, it was investigated whether CR1-like (Complement receptor 1-like, CR1-like) of porcine erythrocyte could bind to the C3b or not.【Method】In this study, the recombinant plasmids of CR1-like(3-6) and CR1-like(8-11) functional domain fragments were constructed first, which were used to establish a yeast two-hybrid detection system. The bait plasmid (recombinant pGBKT7-CR1-like) and capture plasmid (recombinant pGADT7-C3b) were co-transformed into Y2HGold yeast cells. The single deficient SD/-Leu, SD/-Trp and double-deficient SD/-Leu/-Trp (DDO) media were used to strictly screen the co-transformed yeast cells. Then, according to the expression of report factor, the growth of transformants were identified on the double-deficient medium SD/-Leu/-Trp/X-α-Gal (DDO/X) or SD/-Leu/-Trp/X-α-Gal/Aba (DDO/X/A) combined with the color change phenomenon of the colony to comprehensively determine whether CR1-like active fragments and complement C3b bind to each other in yeast cells or not. The CR1-like-C3b binding complex in yeast cells was then separated by immunoprecipitation, and the specificity of the complex was identified by Western blot. 【Result】The co-transformed yeast clones showed normal growth on SD/-Leu, SD/-Trp, DDO and DDO/X, DDO/X/A media with blue color colonies, and this indicated that positive yeast colonies were successfully obtained. The results of PCR reverse identification showed that the co-transformed yeast contained the target genes CR1-like(3-6) and CR1-like(8-11). The C3b gene fragment appeared after the plasmid was digested, indicating that the recombinant plasmid pGBKT7-CR1-like and pGADT7-C3b were successfully co-transformed into yeast cells. In the immunoprecipitation test, the tag antibody c-Myc of the pGBKT7 vector was used to precipitate the fusion protein in yeast cells. Western blot detection with c-Myc as the primary antibody revealed that the fusion protein transformed pGBKT7-CR1-like(3-6) and pGBKT7-CR1-like(8-11) separately showed a specific band at 50 kDa; the yeast fusion protein co-transformed with pGBKT7-CR1-like(3-6) + pGADT7-C3b and pGBKT7-CR1-like(8-11) + pGADT7-C3b showed a specific band at 83 kDa; when the HA monoclonal antibody was used as the primary antibody for Western blot detection, no specific bands appeared in the pGBKT7-CR1-like(3-6) and pGBKT7-CR1-like(8-11) fusion proteins, and only the yeast fusion protein co-transformed in lane 3 and 4 showed a specific band at 83 kD. It showed that there was a complex of CR1-like and C3b in Y2HGold yeast cells. Using CR1-like monoclonal antibody to precipitate the fusion protein in yeast cells, Western blot detection with CR1-like as the primary antibody revealed that the fusion protein transformed with pGBKT7-CR1-like(3-6) and pGBKT7-CR1-like(8-11) separately showed a specific band at 50 kD; the yeast fusion protein co-transformed with pGBKT7-CR1-like(3-6) + pGADT7-C3b and pGBKT7-CR1-like(8-11) + pGADT7-C3b showed a specific band at 83 kD; when the C3 monoclonal antibody was used as the primary antibody for Western blot detection, no specific bands appeared in the pGBKT7-CR1-like(3-6) and pGBKT7-CR1-like(8-11) fusion proteins, lanes 3 and 4 showed that only the co-transformed yeast fusion protein had a specific band at 83 kD. This indicated that there was a biologically active CR1-like and C3b binding complex in Y2HGold yeast cells. The bait plasmid expression products CR1-like(3-6), CR1-like(8-11) fragments and capture plasmid expression products C3b fragment could be combined in yeast cells.【Conclusion】In summary, the recognition ligand for porcine erythrocyte CR1-like to exert immune adhesion function was C3b, which provided an important data basis for the further analysis of the molecular structure of CR1-like functional domain. Keywords:CR1-like;C3b;yeast two-hybrid;immune adherence
PDF (984KB)元数据多维度评价相关文章导出EndNote|Ris|Bibtex收藏本文 本文引用格式 孙雨晨, 贾瑞璞, 范阔海, 孙娜, 孙耀贵, 孙盼盼, 李宏全, 尹伟. 猪I型补体受体与C3b活性片段相互结合的体外检测. 中国农业科学, 2021, 54(19): 4243-4254 doi:10.3864/j.issn.0578-1752.2021.19.018 SUN YuChen, JIA RuiPu, FAN KuoHai, SUN Na, SUN YaoGui, SUN PanPan, LI HongQuan, YIN Wei. Detection of Interaction Between Porcine Type I Complement Receptor and C3b Active Fragment in Vitro. Scientia Acricultura Sinica, 2021, 54(19): 4243-4254 doi:10.3864/j.issn.0578-1752.2021.19.018
用于酵母细胞共转化试验:E.Z.N.A.TM Plasmid Mini Kit I、E.Z.N.A.TM Gel Extraction胶回收试剂盒(Omega,中国),限制性内切酶EcoR I、Xho I、Nco I、BamH I、T4 DNA Ligase连接酶、卡那霉素、氨苄青霉素和50×TAE(Solarbio,中国),10 000×Super Gel Red核酸凝胶染料(Everbrite,美国),5 000 bp DNA Marker(中科瑞泰,中国)。
用于酵母总蛋白提取和Western blot试验:Yeast maker Yeast Transformation System 2、Yeast Protein Extraction Reagent酵母总蛋白提取试剂盒、Yeast Media Set 2 Plus试剂盒(TaKaRa,中国),BeaverBeads™ Protein A/G Immunoprecipitation(海狸生物,中国)。
A—E:共转化了pGBKT7-CR1-like(3-6)和pGADT7-C3b的酵母细胞在SD/-Leu、SD/-Trp、DDO、DDO/X、DDO/X/A培养板的生长情况 Fig. 1Yeast two-hybrid assay for pGBKT7-CR1-like(3-6) and pGADT7-C3b
A-E: The growth of yeasts co-transformed with pGBKT7-CR1-like (3-6) and pGADT7-C3b in SD/-Leu, SD/-Trp, DDO, DDO/X and DDO/X/A culture plates
A—E:共转化了pGBKT7-CR1-like(8-11)和pGADT7-C3b的酵母细胞在SD/-Leu、SD/-Trp、DDO、DDO/X、DDO/X/A培养板的生长情况 Fig. 2Yeast two-hybrid assay for pGBKT7-CR1-like(8-11) and pGADT7-C3b
A-E: The growth of yeasts co-transformed with pGBKT7-CR1-like(8-11) and pGADT7-C3b in SD/-Leu, SD/-Trp, DDO, DDO/X and DDO/X/A culture plates
A:pGBKT7-CR1-like质粒的电泳图:M1:DL2 000 DNA Maker;1:共转pGBKT7-CR1-like(3-6);2:共转pGBKT7-CR1-like(8-11)。B:pGADT7-C3b质粒的电泳图:M2:DL5 000 DNA Marker;1、2:共转pGADT7-C3b Fig. 5Gene identification of recombinant plasmids
A: An electrophoresis image of the pGBKT7-CR1-like plasmid:M1: DL2 000 DNA Marker; 1. Co-conversion pGBKT7-CR1-like;2. Co- transformation of pGBKT7-CR1-like. B: An electrophoresis image of the pGADT7-C3b plasmid: M2: DL5 000 DNA Marker; 1,2. Co-conversion pGADT7-C3b
ZHOUL, YANG ZN, ZHANGM, CHENG GY. Whole-industry chain loss and edible rate of chinese meats Scientia Agricultura Sinica, 2019, 52(51):3934-3942. doi: 10.3864/j.issn.0578-1752.2019.21.020. (in Chinese) [本文引用: 1]
LUO YZ, SUNY, WANGT, QIU HJ. African swine fever: A major threat to the Chinese swine industry Scientia Agricultura Sinica, 2018, 51(21):4177-4187. doi: 10.3864/j.issn.0578-1752.2018.21.016. (in Chinese) [本文引用: 1]
WANGQ. The impact of classical swine fever and African swine fever on pig industry Scientia Agricultura Sinica, 2018, 51(21):4143-4145. doi: 10.3864/j.issn.0578-1752.2018.21.012. (in Chinese) [本文引用: 1]
ZHANG JW, ZHUANG JS, YUAN SS. Molecular epidemiology study on high pathogenic porcine reproductive and respiratory syndrome virus in some regions of China Scientia Agricultura Sinica, 2008, 41(6):1822-1831. doi: 10.3864/j.issn.0578-1752.2008.06.033. (in Chinese) [本文引用: 1]
JIA YH, XU CZ, SUI JY, WU YP, XU BF, CHENY, YANG HL, QIAO CL, CHEN HL. Immunogenicity evaluation of eukaryotic expressing plasmids encoding HA protein of eurasian avian-like H1N1 swine influenza virus Scientia Agricultura Sinica, 2019, 52(5):930-938. doi: 10.3864/j.issn.0578-1752.2019.05.014. (in Chinese) [本文引用: 1]
TAYLOR RP, LINDORFER MA, ATKINSON JP. Clearance of amyloid-beta with bispecific antibody constructs bound to erythrocytes Alzheimer's & Dementia (New York, N Y), 2020, 6(1):e12067. doi: 10.1002/trc2.12067. [本文引用: 1]
ZHU XC, DAI WZ, MAT. Impacts of CR1 genetic variants on cerebrospinal fluid and neuroimaging biomarkers in Alzheimer's disease BMC Medical Genetics, 2020, 21(1):181. doi: 10.1186/s12881-020-01114-x. URL [本文引用: 1]
PRAJAPATI SK, BORLONC, ROVIRA-VALLBONAE, GRUSZCZYKJ, MENANTS, THAM WH, KATTENBERG JH, VILLASISE, DE MEULENAEREK, GAMBOAD, VINETZJ, FUJITAR, XUAN XN, URBANO FERREIRAM, NIÑOC H, PATARROYOM A, SPANAKOSG, KESTENSL, ABBEELEJ V D, ROSANAS-URGELLA. Complement Receptor 1 availability on red blood cell surface modulates Plasmodium vivax invasion of human reticulocytes Scientific Reports, 2019, 9(1):8943. DOI:10.1038/s41598-019-45228-6URL [本文引用: 1]
MCQUAIDF, ROWE JA. Rosetting revisited: A critical look at the evidence for host erythrocyte receptors in Plasmodium falciparum rosetting Parasitology, 2020, 147(1):1-11. DOI:10.1017/S0031182019001288URL [本文引用: 1]
OCHOLA-OYIER LI, WAMAEK, OMEDOI, OGOLAC, MATHARUA, MUSABYIMANA JP, NJOGU FK, MARSHK. Few plasmodium falciparum merozoite ligand and erythrocyte receptor pairs show evidence of balancing selection Infection Genetics & Evolution Journal of Molecular Epidemiology & Evolutionary Genetics in Infectious Diseases, 2019, 69:235-245. doi: 10.1016/j.meegid.2019.02.004. [本文引用: 1]
BARCELLINIW, ZANINONIA, GIANNOTTA JA, FATTIZZOB. New insights in autoimmune hemolytic anemia: From pathogenesis to therapy Journal of Clinical Medicine, 2020, 9(12):3859. doi: 10.3390/jcm9123859. URL [本文引用: 1]
KULIKL, LASKOWSKIJ, RENNERB, WOOLAVERR, ZHANG LA, LYUBCHENKOT, YOU ZY, THURMAN JM, HOLERS VM. Targeting the immune complex-bound complement C3d ligand as a novel therapy for lupus Journal of Immunology, 2019, 203(12):3136-3147. doi: 10.4049/jimmunol.1900620. URL [本文引用: 2]
OLIVEIRA LC, KRETZSCHMAR GC, DOS SANTOSA C M, CAMARGOC M, NISIHARAR M, FARIAST D J, FRANKEA, WITTIGM, SCHMIDTE, BUSCHH, PETZL-ERLERM L, BOLDTA B W. Complement receptor 1 (CR1, CD35) polymorphisms and soluble CR1: A proposed anti-inflammatory role to quench the fire of “fogo selvagem” Pemphigus foliaceus Frontiers in Immunology, 2019, 10:2585. doi: 10.3389/fimmu.2019.02585. URL [本文引用: 1]
SIMMONS KT, MAZZILLI JL, MUELLER-ORTIZS L, DOMOZHIROVA Y, GARCIAC A, ZSIGMONDE M, WETSELR A. Complement receptor 1 (CR1/CD35)-expressing retinal pigment epithelial cells as a potential therapy for age-related macular degeneration Molecular Immunology, 2020, 118:91-98. doi: 10.1016/j.molimm.2019.11.007. URL [本文引用: 1]
CRANEA, BRUBAKER WD, JOHANSSON JU, TRIGUNAITEA, CEBALLOSJ, BRADTB, GLAVIS-BLOOMC, WALLACE TL, TENNER AJ, ROGERSJ. Peripheral complement interactions with amyloid β peptide in Alzheimer's disease: 2. Relationship to amyloid β immunotherapy Alzheimer's & Dementia, 2018, 14(2):243-252. doi: 10.1016/j.jalz.2017.04.015. URL [本文引用: 1]
JOHANSSON JU, BRUBAKER WD, JAVITZH, BERGEN AW, NISHITAD, TRIGUNAITEA, CRANEA, CEBALLOSJ, MASTROENID, TENNER AJ, SABBAGHM, ROGERSJ. Peripheral complement interactions with amyloid β peptide in Alzheimer's disease: Polymorphisms, structure, and function of complement receptor 1 Alzheimer's & Dementia, 2018, 14(11):1438-1449. doi: 10.1016/j.jalz.2018.04.003. URL [本文引用: 1]
NIUS, LIX, ZHANGN, NING GB, ZHANGD, JAHEJOA, MA HL, HAO WF, GAO WW, ZHAO YJ, GAO SM, LI GL, LI JH, YANF, GAO RK, BI YH, HAN LX, TIAN WX. Identification of transcription levels of six immune-related factors in chicken erythrocytes infected with marek’s disease virus Progress in Veterinary Medicine, 2018, 39(12):49-53. doi: 10.3969/j.issn.1007-5038.2018.12.010. (in Chinese) [本文引用: 1]
ZHENG SM, GE YY, MA HW, GAO XL, LIU CN, LV XP. Effect of goose source H5N1 avian influenza virus infection to erythrocyte immune function of duckling Journal of Northeast Agricultural University, 2019, 50(1):45-51. doi: 10.19720/j.cnki.issn.1005-9369.2019.01.006. (in Chinese) [本文引用: 1]
NOMBELAI, LOPEZ-LORIGADOSM, SALVADOR-MIRAM E, PUENTE-MARINS, CHICOV, CIORDIAS, MENAM C, MERCADOL, COLLJ, PEREZL, ORTEGA-VILLAIZANM D M. Integrated transcriptomic and proteomic analysis of red blood cells from rainbow trout challenged with VHSV point towards novel immunomodulant targets Vaccines (Basel), 2019, 7(3):63-91. doi: 10.3390/vaccines7030063. [本文引用: 1]
WEI HW, ZHU NN, WANGH, LIU XQ, DUAN LS, ZHOUX, GUO JR. Influences on wound healing in diabetic mice by blood transfusion treated with improved blood preservation solution Basic & Clinical Medicine, 2020, 40(8):1031-1036. doi: 10.16352/j.issn.1001-6325.2020.08.003. (in Chinese) [本文引用: 1]
SUN YG, YINW, FAN XF, FAN KH, JIANG JB, LI HQ. The cytological observation of immune adherence of porcine erythrocyte Cell Communication & Adhesion, 2012, 19(5/6):79-84. doi: 10.3109/15419061.2012.743999. [本文引用: 1]
ZHANG JJ, WANGC, JIA RP, YINW, FAN KH, SUNN, SUN YG, LI HQ. Study on immune adhesion function and CRl-like expression level of porcine erythrocytes Chinese Veterinary Science, 2018, 48(5):593-604. doi: 10.16656/j.issn.1673-4696.2018.0114. (in Chinese) [本文引用: 1]
ZHANG QQ, LING XY, SUN YC, YINW, FAN KH, SUNN, SUN YG, LI HQ. Screening of membrane-binding proteins of complement receptor 1-like on porcine erythrocytes Chinese Veterinary Science, 2020, 50(1):42-48. doi: 10.16656/j.issn.1673-4696.2019.0213. (in Chinese) [本文引用: 1]
YINW, WANGC, FAN KH, SUNN, SUN YG, LI HQ. In vitro observation: the GFP-E. coli adhering to porcine erythrocytes can be removed by porcine alveolar macrophages Peer J, 2019, 7:e6439. doi: 10.7717/peerj.6439. URL [本文引用: 1]
JIA RP, LING XY, SUN YC, YINW, FAN KH, SUNN, SUN YG, LI HQ. Construction and identification of yeast two-hybrid bait plasmid for porcine erythrocyte CR1-like protein Chinese Journal of Veterinary Medicine, 2020, 56(1):5-9.(in Chinese) [本文引用: 5]
LUCHENAC, ZUAZO-IBARRAJ, ALBERDIE, MATUTEC, CAPETILLO-ZARATEE. Contribution of neurons and glial cells to complement-mediated synapse removal during development, aging and in Alzheimer's disease Mediators of Inflammation, 2018, 2018:2530414. doi: 10.1155/2018/2530414. [本文引用: 1]
SHIL. Clinical observation of serum immunoglobulin, erythrocyte immunity and peripheral blood T lymphocyte subgroup test in the treatment of children with Mycoplasma pneumoniae infection World Latest Medicine Information, 2019, 19(73):195-197. doi: 10.19613/j.cnki.1671-3141.2019.73.127.(in Chinese) [本文引用: 1]
ZHANG ZL, TIAN HW, LI KX, ZHANGR, WANGC, LIQ, LI KQ, NIU XD, ZHANG YR, LIU JZ. Swine Eperythrozoon suis infection Swine Industry Science, 2020, 37(11):99-102.(in Chinese) [本文引用: 1]
BUSUTTIM, DIOMEDI-CAMASSEIF, DONADELLIR, MELEC, EMMAF, VIVARELLIM. Efficacy of eculizumab in coexisting complement C3 glomerulopathy and atypical hemolytic uremic syndrome Kidney International Reports, 2021, 6(2):534-537. doi: 10.1016/j.ekir.2020.10.037. URL [本文引用: 1]
YINW, CUI JY, JIANG JB, ZHAO JX, FAN KH, SUNN, WANG ZW, SUN YG, MA HL, LI HQ. The immune adherence receptor CR1-like existed on porcine erythrocytes membrane Scientific Reports, 2015, 5:13290. doi: 10.1038/srep13290. URL [本文引用: 1]
CHENGJ, JIANG JB, ZHAO JX, WANG ZR, SUN YG, MA HL, FAN KH, YINW, SUNN, WANG ZW, ZHAOX, LI HQ. Cloning and bioinformatics analysis of a full-length cDNA of porcine CR1-like gene Acta Biochimica et Biophysica Sinica, 2014, 46(11):997-1000. doi: 10.1093/abbs/gmu084. URL [本文引用: 1]
GONG WX, YINW, WANGC, FAN KH, SUNN, SUN YG, LI HQ. Study on single nucleotide polymorphism and copy number variation of CR1-like gene in landrace Heilongjiang Animal Science and Veterinary Medicine, 2018(6):113-118, 254. doi: 10.13881/j.cnki.hljxmsy.2018.02.0141. (in Chinese) [本文引用: 1]
XUE YP. The monoclonal antibody of porcine CR1-like and its distribution on erythrocyte membrane [D]. Taigu: Shanxi Agricultural University, 2015. (in Chinese) [本文引用: 1]