Construction Of MTB Hsp16.3 Recombinant Protein And Its Mechanism Of Regulating Macrophage M2-type Polarization

Objective: MTB Hsp16.3 recombinant protein was constructed to verify its role in regulating M2-type polarization of macrophages through CX3CR1.Bioinformatics analysis screened the possible target genes of CX3CR1 and explored the related molecular mechanismMethods:1.Expression of MTB Hsp16.3 recombinant protein: Primers were designed on the basis of bioinformatics analysis according to the HspX gene sequence in NCBI and the recombinant plasmid pGEX-4T-GST-Hsp16.3-His was constructed by homologous recombination technology.The target sequence was amplified by PCR,identified by agarose gel electrophoresis,and recovered by cutting the target band on the gel.The pGEX-4T-GST-Hsp16.3-His plasmid was transformed into BL21 competent cells,and the bacteria were collected after induction by adding IPTG.The bacteria were lysed and disrupted by sonication.After centrifugation,the supernatant and the precipitate were collected for SDS-PAGE analysis.The target fragment recovered from the gel and pMAL-c5 x plasmid were digested with Sac I/BamHI and then ligated with recombinase.The ligation products were transformed into DH5α competent cells,and single colonies were picked for sequencing verification.The expression plasmid pMAL-c5x-MBPHsp16.3-His was transformed into BL21 competent cells to induce protein expression and analyzed by SDS-PAGE gel.b The obtained bacterial protein samples were purified by Ni-column affinity chromatography.The MBP tag was removed by TEV digestion,and the target protein was separated from the MBP tag by Ni column and MBP column.The purification of the target protein was detected by electrophoresis..2.Verification of the role of MTB Hsp16.3 recombinant protein in regulating M2-type polarization of macrophages: Three groups of MTB Hsp16.3 recombinant proteins with concentration gradients were set up to induce mouse bone marrow-derived macrophages respectively.The IFN-γ-induced macrophages were used as the standard control for M1-type polarization,and the IL-4-induced macrophages were used as the standard control for M2-type polarization.The mRNA expression levels of M1-type polarization-related molecules such as TNF-α,iNOS and M2-type polarization-related molecules like IL-10,TGF-β and CX3CR1 were detected by Real-time PCR.Western blot was used to detect the protein expression levels of MHCⅡ,CD206 and CX3CR1.The secretion levels of M1-type polarization marker like IL-6 and M2-type polarization markers such as TGF-β and IL-10 were measured by ELISA.3.The mechanism of MTB Hsp16.3 recombinant protein regulating macrophage M2-type polarization via CX3CR1: using lentivirus with GFP green fluorescent label to infect mouse bone marrow-derived macrophages,and observe the green fluorescence signal intensity of macrophages by fluorescence microscope to determine the optimal infection time and MOI value.Macrophages were treated with PBS as blank control group(cont),infected with lentiviral control vector LV-shRNA-NC as negative control group(shNC),and infected with three lentiviral interference vectors LV-shRNA-CX3CR1 as the experimental group(shRNA1-3);Real-time PCR and Western blot were used to detect the effects of three lentiviruses on the expression of CX3CR1 in macrophages at the mRNA and protein levels,respectively.The lentiviral interference vector with the highest interference efficiency was screened,and the BMDMs infected with the selected lentiviral interference vector were used as the experimental group(shRNA3).MTB Hsp16.3 recombinant protein was added to cont group,shNC group and shCX3CR1 group to stimulate for 24 h and named as Mock group,Scramble group and shRNA3 group,and total cell RNA was extracted for transcriptome sequencing.Comparing the data of shCX3CR1 with cont group and shNC group,the differentially expressed genes of CX3CR1 affecting macrophage polarization were screened out,and GO and KEGG function enrichment analysis of these differentially expressed genes was performed to explore the key signaling pathway of CX3CR1 regulating M2-type polarization of macrophages and and to verify the possible signaling pathways.Results:1.The size of the target fragment obtained by PCR amplification of the recombinant plasmid pGEX-4T-GST-Hsp16.3-His is about 432 bp,which is consistent with the expected length.After using this plasmid as a vector to transform BL21 competent cells,the induced recombinant protein was expressed in a small amount at the expected protein size(43k Da),mainly in the form of inclusion bodies.The pMALc5x-MBP-Hsp16.3-His plasmid was identified by sequencing,and the sequence of the target gene was correct.After transformation into BL21 competent cells,the recombinant protein could be expressed in supernatant at 37°C and 18°C after induction by IPTG.The obtained protein was processed by affinity chromatography column and TEV digestion to obtain the MTB Hsp16.3 target protein.2.MTB Hsp16.3 recombinant protein induces M2-type polarization of mouse bone marrow-derived macrophages and promotes the expression of CX3CR1: The phenotype of mouse bone marrow-derived macrophages stimulated by MTB Hsp16.3 recombinant protein is more consistent with the IL-4 stimulation group.Real-time PCR results indicated that the level of M1-type polarization-related molecules such as TNF-α and iNOS was down-regulated,while the mRNA expression levels of M2-type polarization-related molecules like TGF-β,IL-10 and CX3CR1 were increased,and the differences were statistically significant.(P<0.05);Western blot results showed that the recombinant protein inhibited the protein expression of MHCⅡ,but promoted the protein expression of CD206 and CX3CR1(P<0.05);ELISA results showed that the recombinant protein promoted TGF-β,IL-10 secretion(P<0.05).3.The optimal conditions for lentivirus to silence CX3CR1 on the surface of macrophages were MOI=100 and the infection time was 5d.Three groups of lentiviruses were used to infect macrophages under optimal conditions,and CX3CR1 in the shRNA3-infected group decreased most significantly in both mRNA and protein levels(P<0.05).The quality of transcriptome sequencing results was assessed by Real-time PCR,and the results showed that the Real-time PCR results of the selected 10 genes were consistent with the sequencing results.Using |log2FC|≥0 and p-adjust<0.05 as the screening conditions,a total of 1535 differentially expressed genes were obtained,of which 1420 differentially expressed genes were functionally annotated,and the most significant enriched GO functional items were extracellular matrix,plasma membrane.KEGG pathway enrichment analysis of differentially expressed genes showed that 285 signaling pathways were enriched,of which 63 pathways were significantly enriched.The common downstream pathways of the ten most enriched signaling pathways were JAK/STAT,MAPK and PI3K/AKT.The differentially expressed genes involved in multiple signaling pathways were screened,and the obtained differentially expressed genes constructed a protein interaction network.According to the number of connecting nodes between proteins and bioinformatics analysis,ten potential target genes of CX3CR1 were screened: Lama1/ 2/4/5,Lamb1/2,Lamc1/2 and Itga2/3.To further explore the mechanism of CX3CR1 regulating M2-type polarization of macrophages,Western blot detected the expression levels of key proteins in JAK/STAT,MAPK and PI3K/AKT pathways,and the results showed that the phosphorylation levels of JAK1,JAK2,STAT1,STAT3 and PI3 K increased.Conclusion:1.The recombinant plasmid pMAL-c5x-MBP-Hsp16.3-His was successfully constructed.The expression of soluble recombinant protein MTB Hsp16.3 can be successfully induced by transferring the recombinant plasmid into BL21 competent cells.2.MTB Hsp16.3 can induce M2-type polarization and promote the expression of CX3CR1 in mouse bone marrow-derived macrophages.3.CX3CR1 can regulate the polarization of macrophages to M2 through PI3K/AKT,JAK/STAT and other signaling pathways.