Molecular Cloning And Expression Of Large White Pig CXCL8 And Study Of Its Biological Activity

CXCL8 is a CXC chemokine that control several tissue functions,including cell recruitment and activation such as neutrophils,monocytes,lymphocytes,and the like.Recruitment of circulating neutrophils to the site of infection is the first line in host defense.Such a response should be immediate and robust,yet controlled by CXCL8.However,excessive neutrophil infiltration during inflammation can also lead to excessive inflammation,leading to destruction of host tissue cells.Inflammation is the most important pathological change in the development of most swine diseases,and CXCL8 plays an important role in these diseases.Lagre White Pig is one of the important pig breeds in China,but the study about CXCL8 of the Lagre White Pig is relatively rare.In this study,we cloned the CXCL8 gene of Lagre White Pig,and the CXCL8 and CXCL8mutant(G58P)protein were expressed in soluble,and their biological activity had been verified.Our study provides a basis for further study of CXCL8's function and mechanism and the development of targeted drugs that inhibit swine inflammation.The main research contents are as follows:1 Cloning,Expression and Purification of CXCL8 and G58 P mutant.The mRNA of lung tissue of Large White Pig was extracted and the CXCL8 gene was amplified by RT-PCR.After sequencing,the gene was cloned into p GEX-6p-1 vector to obtain the recombinant plasmid p GEX-PCXCL8 After expression conditions optimized,the exogenous gene expression in the supernatant.SDS-PAGE and Western Blot showed that the protein was purified well through protein purification system AKTA prime 10.The CXCL8 58 th glycine(G)was mutated to proline(P),and the CXCL8 mutant gene G58 P was synthesized according to the corresponding base sequence,and then subcloned into p GEX-6p-1 vector through DNA recombination.Expression and purification methods are the same as CXCL8.2 CXCL8 in vivo activity validationBALB/c mice were inoculated intranasally with 25 ?L of different concentrations of PCXCL8 recombinant protein(4mg/m L,400?g/m L,40?g/m L,4 ?g/m L),and control mice were inoculated with the same volume and concentrations of GST protein,and the blank group were inoculated with the same volume of normal saline.After 12 hours,all the mice were sacrificed.Take the lungs.The biological activity of the recombinant protein was assessed by histopathological observation,BALF neutrophil count,and MPOactivity detection in lung tissue.Histopathological observation showed that mice of different concentrations of PCXCL were pneumonia,and 4mg/m L GST group shows mild inflammation,and the rest of the GST group and saline control group were no significant abnormalities;PCXCL8 experimental group's MPO activity were significantly higher than the control Group as well as the normal saline group.The number of BALF neutrophils in 4mg/m L PCXCL8 group was much higher than that in GST group.3 G58 P in vivo activity validationBALB/c mice were inoculated intranasally with G58P(4mg/m L,25?L)and set the PCXCL8 positive control group,PCXCL8 and G58 P mixed group,normal saline control group,untreated blank control group at the same time.After 12 hours,all the mice were sacrificed.Take the lungs.The biological activity of G58 P was assessed by histopathological observation,BALF neutrophil count,and MPO activity in lung tissue.Histopathological observation showed that the G58 P experimental group,mixed group and PCXCL8 control group showed pneumonia characteristics while saline control group and blank control group was no significant abnormalities;The MPO activity of G58 P experimental group,mixed group and PCXCL8 control group were no significant difference,but were significantly higher than those in the normal saline control group and the blank control group;Similarly,The number of BALF neutrophils in the G58 P experimental group,the mixed group and the PCXCL8 control group were no significantly difference,but were significantly higher than those in the saline control group and the blank control group.Conclusion: Soluble expression of recombinant PCXCL8 and G58 P was achieved by using prokaryotic expression vector p GEX-6p-1.Both PCXCL8 and G58 P have good biological activity.The PCXCL8 58 th amino acid G-P mutation has no significant effect on its biological activity.