Study On Porcine Erythrocyte Immune Adherence And Its Receptors

Veterinary studies have shown that erythrocytes of non-primate mammals have immunoadhesive functions.However,presently,only studies on ECR1 protein in primates are clear while those on the protein characteristics of immune adherent receptors on erythrocytes of other animals,as well as the distribution on erythrocyte membranes,the coding gene structure and its chromosome localization,and the mechanisms of immune function are almost non-existent.The porcine erythrocyte immune adhesion molecule complement receptor 1(CR1)-like was isolated and identified in a previous study,but a number of basic questions about the mechanism of porcine erythrocyte immune response need to be addressed in further studies.For example,the physiological factors that affect CR1-like immune activity,the anchoring characteristics of CR-1 on the surface of erythrocytes,the CR1-like sequence homology conservation at the species level,and the existence of porcine erythrocyte CR1-like molecular polymorphisms are yet to be investigated.Based on the above background and pending scientific question,in this study,the porcine CR1-like polyclonal antibody was prepared using the CR1-like molecule as a study target.The scaffolding region of the CRl-like connecting membrane skeleton protein and the molecular basis of the interaction between the CR1-like protein and porcine erythrocyte membrane were analyzed in detail.The role of ATP in regulating CR1-like immune adhesion was investigated using flow cytometry,fluorescence,immunocytochemistry,and immunosuppression techniques,using the correlation between ATP and porcine erythrocyte CR1-like protein as the study target.This investigation provided preliminary data for further elucidation of the molecular mechanisms of CR1-like-mediated immune function in porcine erythrocytes.The genetic basis of the CR1-like immunoadhesion function of porcine erythrocytes was established using gene library construction and single nucleotide polymorphism(SNP)screening,to provide scientific data and establish a theoretical foundation for further development of CR1-like gene polymorphisms of porcine erythrocytes for clinical applications.Experimental results showed:1.The specific antibody against porcine CR1-like membrane binding peptide with immunological activity,which was successfully prepared,provided a research tool for further studies of the molecular mechanisms of porcine ECR1-like molecular immunological function.The expression system of CR1-like protein fragments of the porcine erythrocyte CR1-like protein activity was successfully established,and recombinant Pichia pastoris that stably expressed the porcine erythrocyte CR1-like protein was successfully obtained.The expression amount of the recombinant protein CCP was 0.945 mg/mL.In vitro activity studies have shown that the recombinant protein CCP exhibits functional homology with the native CR1-like molecule,and has complement-binding activity and immune-like adhesion functions.These results provide the basis for technical methods and theoretical data for further in-depth studies of the mechanism of action of erythrocyte immune molecules.2.The molecular basis of the effects of CR1-like protein distribution on immune function was studied in-depth using a cellular in situ fluorescence staining method,geometric position evaluation,and immunoprecipitation.Firstly,results of the immunofluorescence cytochemical staining showed that the specific fluorescence site distribution of both the CR1-like and Fas-associated phosphatase-1(FAP-1)molecules in the porcine erythrocyte membrane skeleton were similar.Secondly,the sum of squares of the differences in range distance of 253 typical positive red blood cells was 0.2224,which showed that the difference in the distance of the CR1-like and FAP-1 molecules was 0.This indicates that the distribution of the CR1-like and FAP-1 molecules was consistent with their localization characteristics.Thirdly,the interaction between the CR1-like and FAP-1 molecules was further analyzed using immunoprecipitation.Total protein samples were incubated with the CR1-like protein and then analyzed using western blotting with the FAP-1 monoclonal antibody.The FAP-1 molecule was clearly observed in the gel.The above results indicated that the porcine erythrocyte CR1-like protein did not bind directly to the erythrocyte membrane protein,but was distributed on its surface by FAP-1 protein riveting.3.The ATP concentration decreased when the CR1-like protein was incubated with a 1:1600 dilution of CR1-McAb.However,the 1:1600 dilution is not close to the optimal binding ratio of the CR1-like protein and the antibody and,therefore,the binding intensity between them was lower than that of other dilutions.In addition,the ATP consumption at 1:1600 was also significantly lower than that at other dilutions.Thus,the pig erythrocyte ATP was consumed during the binding of the CR1-like protein and antibodies.The immunoactivity of the CR1-like protein can also be initially inferred based on the ATP concentration at different dilutions.The stronger the CR1-like immunoactivity,the higher the ATP consumption is.There is a significant correlation between CR1-like protein and ATP.When the concentration of ATP was increased to 10-14,10-13,10-12,10-10 and 10-8 mol/mL,no statistical differences in the expression level of the CR1-like membrane was observed between the groups.There are no nuclei in mature pig erythrocytes because they are mammals.The CR1-like protein expression level on the surface of erythrocytes is normal,constant,and is not altered by changes in the ATP concentration.Furthermore,the effect of ATP on CR1-like immunoadhesion was examined based on the adhesion fluorescence intensity and efficiency using flow cytometry and fluorescence immunocytochemistry.The results showed that when the ATP concentration was increased to 10-145 10-13,10-12,10-10,and 10-8 mol/mL,the number of CRl-like immunoadherent fluorescent particles and the efficiency of adhesion bacteria were also increased.These results also showed that regardless of adhesion fluorescence intensity or adhesion bacteria efficiency,the number of CR1-like adherent particles was highest when the ATP treatment concentration was 10-10 mol/mL.Subsequent increases in ATP treatment concentration to 10-8 mol/mL did not induce any changes in the number of CRl-like adherent particles.Therefore,it can be seen that ATP has a dose-related effect on the relative CR1-like immune binding activity at a certain concentration range.The higher the ATP concentration in the adhesion system,the stronger the CR1-like immunoreactivity was.4,Based on the above study results,the CR1-like gene library was further constructed,and candidate SNPs were screened.The experimental results reveal that the CR1-like gene libraries of 41 Changbai pigs were successfully constructed in this study.The coverage of the library was more than 95%,and 98%of the library sequence covered more than 97%.In comparison with the reference genome,159 SNP sites were screened against the CR1-like genome.In addition,the results of this experiment suggest that the position base types of 74513501 and 74515069 should be A and T,respectively.The experimental results were sorted into three reads and uploaded into GenBank.Based on the results,the CR1-like library satisfied the requirements of subsequent experiments,which provided scientific data as a basis for further studies of CR1-like polymorphism in the late stage by the research group.Moreover,it provides a theoretical foundation for the molecular mechanism studies of the CR1-like protein and its disease correlation.