Prokaryotic Expression And Antibody Preparation Of FCGRT And PIGR Genes In Bactrian Camel

In mammals,the immunoglobulin receptor plays an important role in immune defense,while the Fc receptor??chain?responsible for the transport of immunoglobulin G?IgG?and the polymeric immunoglobulin receptor?pIgR?responsible for the transport of immunoglobulin A?IgA?and immunoglobulin M?IgM?are encoded by FCGRT and PIGR genes respectively.In order to prepare polyclonal antibodies against FcRn and pIgR of Bactrian camel,and provide basis for further study on the distribution of immunoglobulin receptor on different mucosal surfaces and the relationship between mucosal immune response in the later period of our laboratory.This study consists of the following two aspects:?1?Firstly,the coding regions were selected from FCGRT and PIGR gene sequences of Bactrian camel included in NCBI,and then the extracellular sequences without signal peptide were intercepted by transmembrane structure and signal peptide prediction analysis,and then the recombinant plasmids pET-28a-FcRn and pET-28a-pIgR were constructed after the prediction of enzyme cutting site;then,they were transferred into the receptive cells BL21?DE3?and Rosetta?DE3?respectively,and then they were transformed into the receptive cells BL21?DE3?and Rosetta?DE3?by1.0 mmol?L^-1 SDS-PAGE was used to detect the expression of IPTG.Then,the IPTG concentrations of 0.1 mmol?L^-1,0.3 mmol?L^-1,0.5 mmol?L^-1,0.7 mmol?L^-1,0.9 mmol?L^-1 and 1.0 mmol?L^-1 were induced for 6 hours respectively,and the SDS-PAGE was used to detect the samples;the optimal IPTG concentrations were induced for 1 h,2 h,3 h,4 h,5 h,6 h,7 h,8 h,9 h,10 h respectively.At last,the LB culture medium was transferred to 2×YT culture medium?including Kan⁺,1:100?.When the OD₆₀₀ value reached 0.6⁰.8,the expression was induced by the best IPTG concentration and the best induction time.After three times of bacteria collection,washing and repeated freezing and thawing,the supernatant and precipitate were collected by centrifugation until the liquid was clear by ice bath ultrasound Samples were tested by SDS-PAGE.The results showed that the expression of FcRn and pIgR was successful in the form of inclusion body,the size was the same as expected,34 kDa and 71 kDa,respectively;the best IPTG induction concentration was 0.3 mmol?L^-1 and 0.1 mmol?L^-1,the best induction time was 6 h and 7 h.?2?Urea binding for inclusion rich precipitation Buffer was dissolved,purified with Ni column,the eluate was collected,and the elution curve was drawn;then,the protein content of the target protein was calculated by the standard curve of bovine serum albumin,and the sample was prepared for SDS-PAGE detection;then,the rabbits were fully emulsified with Freund's complete adjuvant and Freund's incomplete adjuvant respectively,and immunized with subcutaneous injection of back,scapula and popliteal lymph nodes for the fourth time.After 6 days of immunization,blood was collected from the heart and serum was separated,and the appropriate amount was detected by indirect ELISA and Western blotting.The results showed that the peak values of FcRn and pIgR were 4.400 mg·mL^-1 and 1.804 mg·mL^-1 in the second tube,respectively,and the purified recombinant protein was high purity,without heteroprotein,with the size of 34 kDa and 71 kDa,respectively.The titer of serum was 1:32000 and 1:64000 by indirect ELISA,respectively.The results of blotting showed that the Rabbit anti Bactrian camel FcRn and pIgR antibodies could specifically bind to the recombinant protein,that is to say,they had reactivity.Therefore,the polyclonal antibodies against FcRn and pIgR of Bactrian camel have been successfully prepared with high efficiency and good specificity in order to lay a foundation for the later study of the relationship between the distribution of immunoglobulin receptor on different mucosal surfaces and mucosal immune response in our laboratory.