Preparation Of Swine FGL1 Monoclonal Antibody And Establishment Of A Double-antibody Sandwich ELISA Method

Fibrinogen-like protein 1?FGL1?,also known as Hepatocyte-derived fibrinogen-related protein 1?HFREP1?or Hepatocyte proliferative factor?Hepassocin?,is a liver factor with mitogen activity in hepatocytes.It was originally discovered as a transcription product overexpressed in hepatocellular carcinoma cells.The protein,angiogenin-related proteins,and fibrinogen-like protein 2 belong to the fibrinogen superfamily.Under normal physiological conditions,FGL1 is mainly secreted by hepatocytes and is involved in mitosis and metabolism of hepatocytes.Compared with normal hepatocytes,m RNA and protein expression levels of FGL1 were decreased or absent in hepatocellular carcinoma cells.Studies from humans and mice have shown that host cell co-inhibitory molecule expression up-regulation is one of the important causes of persistent infection,tumor and autoimmune disease.Lymphocyte Activation Gene 3?LAG3?is a class of co-inhibitory molecules mainly expressed on the surface of activated T cells.Studies have shown that FGL1 is the main inhibitory ligand of LAG3,and the interaction between FGL1 and LAG3 can inhibit the anti-tumor effect of T cells in vivo and in vitro,and FGL1 silencing can promote the anti-tumor effect of T cells in the mouse model,thus revealing a new immune escape mechanism.In order to study the role of swine FGL1 in the pathogenesis of immunosuppressive swine disease and to accumulate materials for related studies,total RNA was extracted from swine liver tissue and the FGL1 gene was amplified by RT-PCR.Then the purified recombinant protein of swine FGL1 was obtained by prokaryotic expression.The mouse monoclonal antibody and rabbit polyclonal antibody against swine FGL1 were prepared by immunizing mice and rabbits,respectively.Based on the identification of antibodies,a double-antibody sandwich ELISA method was established to detect swine FGL1.The results of this study will accumulate data and provide a basis for the research of FGL1 in swine immune suppressive diseases.The contents and results of this study are as follows:?1?Total RNA of swine fresh liver tissue was extracted,and the FGL1 gene fragment was amplified by RT-PCR.The target gene was inserted into p QE-30 vector to construct a prokaryotic expression vector?p QE-30-FGL1?.After IPTG induction,the prokaryotic expressed swine FGL1 was obtained.The obtained protein was identified by SDS-PAGE and Western blot methods.Then a large number of FGL1 proteins were expressed and purified.?2?Balb/C mice were immunized with the purified swine FGL1.After cell fusion and subcloning,two strains of hybrid tumor cells which could secrete swine FGL1 antibody stably were obtained and the hybrid tumor cells were named 2D7 and 4G7,respectively.The antibody subclass identification showed that the antibodies secreted by the two strains were Ig G1.Western blot and IFA identification results showed that 4G7 could be used for both Western blot and IFA detection of swine FGL1.Also,polyclonal antibodies against swine FGL1 were obtained by immunizing rabbits with purified swine FGL1.?3?Using the obtained mouse monoclonal antibody and rabbit polyclonal antibody against swine FGL1,a double-antibody sandwich ELISA method for detecting swine FGL1was established with the monoclonal antibody as the capture antibody and the rabbit polyclonal antibody as the detection antibody.A series of conditions of the method were also optimized.The results showed that the optimal coating concentration of the captured antibody was 1?g per well and was coated overnight at 4?.The samples to be tested were added and incubated for 1 h at 37?,then the rabbit polyclonal antibody against swine FGL1diluted with 1:1,000 was added and incubated at 37?for 1 h.After washing,1:3,000diluted HRP-labeled equine anti-rabbit Ig G was added and incubated at 37?for 1 h.Finally,TMB substrate was added for color development at room temperature for 20 min away from light,and OD_450nmwas detected by a microplate reader.In conclusion,swine FGL1 protein was successfully expressed in this study.Specific monoclonal antibody and rabbit polyclonal antibody against porcine FGL1 were prepared.Then a double-antibody sandwich ELISA for detection of swine FGL1was established which can provide technical support for the follow-up study on the role of swine FGL1 in immunosuppressive diseases in swine.