Concentration And Purification Of Red Algae Lectin GRFT By GEM And Its Anti-PRRSV Activity Study

In recent years,with the rapid development of bioengineering technology and fermentation technology,the exploration and application of protein are increasing.The purification process gets more complicated and has become the bottleneck in the development of biotechnology.Developing a method that is easy to operate,low in cost is crucial for protein purification.Gram-positive bacterial enhancer matrix surface display system is a novel antigen display system that is developed from the surface display system of gram-positive bacteria Lactococcus tactics.This technique can be used to display the exogenous protein expressed with PA protein anchors together on the surface of GEM particles,so as to achieve the purpose of protein purification.GRFT is an anti-HIV protein derived from Griffithsia sp..This antiviral protein consists of 121 amino acids and exists naturally as a stable dimer.Molecular weight of each single GRFT subunit has three nearly equivalent glycan-binding sites.Because of its characteristics of high efficiency,wide spectrum,stability and safety,GRFT has become a candidate of antiviral drugs.Therefore,this experiment expressed the GRFT-PA fusion protein,aiming to use the specific binding between GEM and PA to purify the GRFT,and make use of the binding of GRFT and virus to verify its activity against PRRSV.In conclusion,our experiment provides a feasible idea for the optimization of protein purification process.The main contents are as follows:Test I preparation of GRFT-PA and display GRFT-PA by GEM particlesExpression and identification of fusion protein GRFT-PA.Firstly,the recombinant plasmid containing PA gene was used as the template for PCR amplification.And the target fragments were recovered and cloned into PMD-19T vector.Then 19T-PA which is identified correct and pET-32a(+)vector,19T-GRFT were enzyme digestion,enzyme ligation,and transformed to DH5? competent cells to obtain the expression vector pET-32a(+)-GRFT-PA,which was transformed into BL-21(DE3)to induce expression.Finally,SDS-PAGE and Western-blot were used to identify the target protein,and the results showed that the recombinant protein was expressed.The target protein was displayed on the surface of GEM particles.The crude extracts containing different volumes of the fusion protein GRFT-PA were mixed with 1 U GEM particle at room temperature for 5 min,and the precipitation were collected by centrifugation.The results showed that GRFT-PA could be successfully displayed on the surface of GEM particles,but not complete.GRFT-PA would appear in the supernatant when 1 U GEM particle was combined with over 120 g recombinant protein.Test ? The activity of purified protein GRFT-PA against PRRSVGRFT-PA was prepared and then combined with GEM to obtain GEM-PA-GRFT complex.The biological activity of GRFT-PA was detected by ELISA,suggesting that the target protein prepared in this study had the expected glycosylation activity.To verify its activity against PRRSV,we make research on Marc-145 cells.Its anti-PRRSV ability was testified by indirect immunofluorescence and Western-blot,and the comparison of anti-PRRSV ability between GEM-PA-GRFT and GRFT was detected by RT-qPCR.The results showed that GEM-PA-GRFT complex could inhibit the infection of PRRSV to marc-145 cells,and GEM-PA-GRFT has stronger ability of anti-PRRSV than GRFT.In addition,GEM-PA-GRFT was added at different time points to explore its effect on the inhibition of PRRSV.he results showed that its anti-PRRSV effect might be mainly in the initial stage of PRRSV invasion.