Detection And Identification Of Membrane-binding Proteins Of Complement Receptor 1-like On Porcine Erythrocytes

Objective:In order to provide theoretical data for exploring the molecular basis of CR1-like distribution on erythrocyte membranes and the research about "the molecular mechanism of the distribution of porcine erythrocyte CR1-like affecting the function of immune adhesion",we identified CR1-like membrane-binding proteins on the surface of porcine erythrocytes.Methods:This study was conducted on healthy adult white pigs.The experimental methods are as follows:1)Using CR1-like immunomagnetic beads prepared by immunoprecipitation,CR1-like complex was isolated and purified from porcine erythrocyte membrane proteins and identified by SDS-PAGE and Western blot.2)CR1-like complex was identified by mass spectrometry and the datas obtained were analyzed by bioinformatics methods from subcellular localization,molecular function annotation and domain.And the candidate protein was determined according to the bioinformatics results.3)Erythrocyte membrane protein band 4.1 in erythrocyte membrane proteins and CR1-like complex was detected by Western blot.4)The primers were designed using the CDS sequence of EPB41 gene as the template,and the c DNA of porcine blood was amplified by PCR and detected by agarose gel electrophoresis.The target band was sequenced and synthesized.5)The recombinant plasmids CR1-like and EPB41 were constructed and were transferred into HEK293 t cells by transient co-transfection technology.After 24 hours of cell culture,protein samples were prepared and detected by immunoprecipitation and Western blot to confirm the interaction between CR1-like and Erythrocyte membrane protein band 4.1.Results: 1)The results of reducing SDS-PAGE of CR1-like complex showed that there were 3 distinct protein bands,the molecular weight of which were 163.178±1.518 k Da,49.921±0.378 k Da,and22.088±0.049 k Da respectively.The molecular weight of the proteins were 284.751±14.347 k Da and199.280±0.947 k Da respectively under non-reductive conditions.2)117 candidate proteins were screened out by mass spectrometry from CR1-like complex.After bioinformatics analysis,erythrocyte membrane protein band 4.1 was determined as the candidate protein for follow-up verification because of the highest score.3)Western blot analysis of erythrocyte membrane total proteins and CR1-like complex revealed the molecular weight of 83.49 kda and 64.21 kda,respectively.4)As expected,a single gene fragment with a size of 228 bp was detected by agarose gel electrophoresis.5)HEK293t cells transfected with CR1-like and EPB41 recombinant plasmid were detected by Western blot,and the results showed that there was an interaction between CR1-like and Erythrocyte membrane protein band 4.1.Conclusions: 1)The method of immunomagnetic beads precipitation for CR1-like was established.2)And the experiment confirmed that CR1-like binding with erythrocyte membrane protein band 4.1 was distributed on the surface of porcine Erythrocyte membrane.