Expression and characterization of Gag protein of HIV-1 （CN） in Pichia pastoris

To express the core protein of HIV-1 of Chinese prevalent strata （HIV-1[CN]） in Pichia pastoris, the fulllength gag gene was inserted into the secretory expression vector pHILS1. Linearized recombinant plasmid pHILGAG by Sal I was electrotransformed into the yeast strain GS115, and the yeast transformants were identified by PCR. To induce the interest protein to be expressed, the PCR positive transformants were inoculated in the medium of BMGY and BMMY, mRNA of the strain was detected by RT-PCR, and the expressed protein was analyzed by SDS-PAGE, Western blotting and thin layer scanning. mRNA （1.3 kb） was amplified by RT-PCR. SDS-PAGE and Western blotting analysis showed that the molecular mass of the expressed protein was 55 kDa, which was similar to the expected value, and the expressed protein could react with McAb to HIV-1 p24. Thin layer scanning analysis demonstrated that the whole amount of the expressed protein was approximately 13% of the soluble protein in the supernatant. The recombinant yeast had good genetic stability. The optimal expression conditions of the engineering yeast were as follows： BMMY medium, 80%-90% of dissolved oxygen, 1% methanol, and 3-day-cultivation course. Gag proteins were expressed under the optimal expression condition and purified via gel filtration chromatography. The purity of the interest protein was up to 85%. After the purified proteins were inoculated into BALB/c mice, the anti-HIV-1 antibodies in the immunized mice could be detected by Western blotting.     

Expression and identification of type 1 diabetes associated autoantigen IA-2

Objectives To obtain prokaryotic expressed IA-2 recombinant protein and to identify its immunological activity.Methods The complimentary DNA (cDNA) coding for the intracytoplasmic part of IA-2 (IA-2ic) was amplified from human fetal brain RNA, and was subcloned into the PinPoint Xa-1 T vector to construct recombinant expression plasmid, and was then expressed in E. coli JM109 cells as a fusion protein with a biotinylated peptide sequence at the aminoterminus. The biotinylated fusion protein was then purified by affinity chromatography and was subsequently dialyzed. Finally, its immunogenicity was evaluated by enzyme linked immunosorbent assay (ELISA).Results The purified IA-2ic fusion protein resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as a single Coomassie brilliant blue stained band with a molecular weight of 59 kDa and its immunogenicity was confirmed by ELISA.Conclusions E. coli expressed IA-2ic fusion protein has immunological activity. It can be used for detection of IA-2 autoantibodies (IA-2A) and for further studies on type 1 diabetes in future,     

Construction of Prokaryotic Expression Plasmid of Fusion Protein Including Porin A and Porin B of Neisseria Gonorrhoeae and Its Expression in E. coli

In order to provide a rational research basis for clinical detection and genetic engineering vaccine, plasmid pET-28a ( ) encoding both Porin gene PIA and PIB of Neisseria gonorrhoeae was constructed and a fusion protein in E. coli DE3 expressed. The fragments of PIA and PIB gene of Neisseria gonorrhoeae were amplified and cloned into prokaryotic expression plasmid pET-28a ( ) with double restriction endonuclease cut to construct recombinant pET-PIB-PIA. The recombinant was verified with restriction endonuclease and sequenced and transformed into E. coli DE3 to express the fusion protein PIB-PIA after induced with IPTG. The results showed PIA-PIB fusion DNA fragment was proved correct through sequencing. A 67 kD (1 kD=0. 992 1 ku) fusion protein had been detected by SDS-PAGE. It was concluded that the fusion protein was successively expressed.     

Blockage of PPAR8 increases the expression of-inflammatory factors in 3T3-L1 cells stimulated with TNFα

Objective： To investigate the role of peroxisome proliferator-activated receptors δ （PPARδ） in inflammatory reaction and its possible mechanism in adipocyte. Methods：Lentivirus-mediated RNA interference （RNAi） was used to block the expression of PPARδ in 3T3-L1 cells. In order to induce inflammation in 3T3-L1, cells were stimulated with tumor necrosis factor-α（TNFα, 20 ng/ml） for 4 h. The expression of PPARδ, nuclear factor κB （NFκB） and C reactive protein （CRP） were determined by Western blot analysis. Results：The expression of PPARδ was reduced by 80% after RNAi. Blockage of PPARδ promoted the expression of CRP and NFκB in cells stimulated with TNFα but had no effect on normal cells. Conclusion： PPARδ is involved in inflammatory reaction in adipocyte. Blockage of PPARδ can promote the inflammation mediated by inflammatory factors and increase the expression of NFκB and CRP in 3T3-L1 cells stimulated with TNFα.     

Expression and characterization of Gag protein of HIV-1（CN） in Pichia pastoris

To express the core protein of HIV-1 of Chinese prevalent strain （HIV-1 （CN）） in Pichia pastoris, the fulllength gag gene was inserted into the secretory expression vector pHILS1. Linearized recombinant plasmid pHILGAG by Sail was electrotransformed into the yeast strain GS115, and the yeast transformants were identified by PCR. To induce the interest protein to be expressed, the PCR positive transformants were inoculated in the medium of BMGY and BMMY, mRNA of the strain was detected by RT-PCR, and the expressed protein was analyzed by SDS-PAGE, Western blotting and thin layer scanning. mRNA （1.3 kb） was amplified by RT-PCR. SDS-PAGE and Western blotting analysis showed that the molecular mass of the expressed protein was 55 kD, which was similar to the expected value, and the expressed protein could react with McAb to HIV-1 p24. Thin layer scanning analysis demonstrated that the whole amount of the expressed protein was approximately 13 % of the soluble protein in the supernatant. The recombinant yeast had good genetic stability. The optimal expression conditions of the engineering yeast were as follows： BMMY medium, 80-90% of dissolved oxygen, 1% methanol, and 3-day-cultivation course. Gag proteins were expressed under the optimal expression condition and purified via gel filtration chromatography. The purity of the interest protein was up to 85 %. After the purified proteins were inoculated into BALB/c mice, the anti-HIV-1 antibodies in the immunized mice could be detected by Western blotting.     

Construction of Prokaryotic Expression Plasmid of mtrC Protein of Neisseria gonorrhoeae and Its Expression in E. Coli

Summary： In order to provide a rational research basis for detection of resistance of Neisseria gonorrhoeae to antimierobial hydrophobie agents and study on the resistant mechanism of multiple transferable resistance （mtr） efflux system, plasmid pET-28a（＋） encoding mtrC gene was constructed and the related target protein was expressed in Escherichia colt （E. cold DE3. The fragments of mtrC gene of Neisseria gonorrhoeae from the standard strains were amplified and cloned into prokaryotic expression plasmid pET-28a（＋） with restriction endonuelease to construct recombinant pET-mtrC which was verified by restriction endonuelease and DNA sequencing. The recom- binant was transformed into E. coli DE3 to express the protein mtrC induced by IPTG. The results showed mtrC DNA fragment was proved correct through restriction endonuelease and DNA sequencing. hs sequence was 99.5 % homologus to that published on GeneBank （U14993）. A 48.5 kD fusion protein which was induced by IPTG was detected by SDS-PAGE. It was concluded that the construction of prokaryotic expression plasmid of mtrC protein of Neisseria gonorrhoeae was correct and the fusion protein was successively expressed in E. coli.     

Stable Expression of Hantavirus H8205 Strain G1/IL-2 Gene and Immune Protection of the Fusion Gene

To explore the feasibility of stable expression of Hantavirus H8205 strain G1 segment and human IL-2 fusion gene in Vero cells, and to examine the immune protection effects on mice vaccinated with this recombinant eukaryotic expression vector containing Hantavirus G1 gene and IL-2 gene. With the help of lipofectamine, the Vero cells were transfected with pcDNA3.1/HisB-IL-2-G1 and the positive cells were selected by G418. IFAT and SDS-PAGE elec- trophoresis were used to determine the stable transfection and expression of recombinant protein. Each mouse was inoculated with plasmids intramuscularly (i.m.) three times, 2 boosts were given at 2-week intervals, serum anti-hantavirus antibodies were detected by ELISA and neutralizing antibod- ies (NAb) were detected by Plaque Reduction Neutralization Test. The fusion protein expressed in Vero cells was 78 kD, corresponding to the estimated molecular size. The neutralizing antibody titers of mice with pcDNA3.1/HisB-IL-2-G1 were 1:20-1:80. IL-2/G1 fusion gene could be transferred in Vero cells and stably express the fusion protein. Specific humeral immune responses in mice can be induced with the recombinant eukaryotic expression vector containing the fusion gene, which lays the foundation for further development of therapeutic HTNV vaccine.     

Construction,Expression and in vitro Biological Effects of Idiotype Ig Fab Fragment of B-Chronic Lymphocytic Leukemia

The purpose of this study was to construct expression vectors of idiotype （Id） SmIg in patients with B-chronic lymphocytic leukemia and to express them in E.coli to obtain recombinant Id, and to investigate the effect of the protein on the proliferation and secretion of IL-2 and IFN-γ of stimulated peripheral blood mononuclear cells （PBMC） in vitro. Light chain gene and Fd fragment of heavy chain gene were inserted into fd-tet-DOG2 vector to construct fd-tet-DOG2-Fab. Fab gene was further cloned into expression vector pHEN2 to construct the soluble expression vector pHEN2-Fab. After induction by IPTG, Fab protein was purified by Ni-NTA-chromatography. MTT was used to determine the effects of purified protein on the proliferation of stimulated PBMC in vitro and the concentrations of IL-2 and IFN-γ in the culture supernatants were detected by ELISA. The results showed that recombinant pHEN2-Fab expression vector was constructed successfully. Fab protein was expressed in positive clone after induced by IPTG and two specific bands at 24-25 kD position were observed by SDS-PAGE electrophoresis. Proliferation of PBMC could be induced by purified Fab and the concentrations of IL-2 and IFN-γ in culture supernatants were increased significantly af- ter induction. It was suggested that the expression vector of SmIg Fab fragment was constructed suc- cessfully, and expressed and secreted from E. coli. The Fab protein could induce proliferation of PBMC and promote secretion of IL-2 and IFN-γ.     

Blockage of PPARδ increases the expression of inflammatory factors in 3T3-L1 cells stimulated with TNFα

Objective: To investigate the role of peroxisome proliferator-activated receptors δ (PPARδ) in inflammatory reaction and its possible mechanism in adipocyte. Methods :Lentivirus-mediated RNA interference (RNAi) was used to block the expression of PPARS in 3T3-L1 cells. In order to induce inflammation in 3T3-L1, cells were stimulated with tumor necrosis factor-α(TNFα, 20 ng/ml) for 4 h. The expression of PPAR8, nuclear factor κB (NFκB) and C reactive protein (CRP) were determined by Western blot analysis. Results:The expression of PPARδ was reduced by 80% after RNAi. Blockage of PPARδ promoted the expression of CRP and NFκB in cells stimulated with TNFα, but had no effect on normal cells. Conclusion: PPARδ is involved in inflammatory reaction in adipocyte. Blockage of PPARδ can promote the inflammation mediated by inflammatory factors and increase the expression of NFκB and CRP in 3T3-L1 cells stimulated with TNFα.     

Optimization of the construction of recombinant plasmids PPARγ- pSUPER-EGFP for RNA interference

Objective：To construct and identify the recombinant plasmids PPARγ-pSUPER-EGFP for RNA interference. Methods： The pSUPER-EGFP vectors were used to transcribe functional short interfering RNA （siRNA）. Four pairs of 64 nt PPARγ siRNA encoding sequences were inserted into the downstream of the H1 promoter. The recombinant plasmids were confirmed by double digestion with the enzymes and sequencing. Western blotting was used to examine the silencing effect of PPARγ gene in RAW264.7 cells. Following procedures were used to optimize the experiments： the oligonucleotides were incubated 5 min at 95 C and cooled automatically in boiled water bath to anneal, and then phosphorylated oligonucleotides, pSUPER-EGFP plasmids was digested with Bgl Ⅱ and Hind Ⅲ , and the product was ligated into digested pSUPER-EGFP plasmids, and transforming the ligation products followed by screening and identifying positive clones. Results ：Four kinds of positive clones producing 285 bp fragments were selected. Sequencing further proved their correctness. Four recombinant plasmids containing corresponding PPARγ gene-specific target sequences induced the silencing of its target gene more or less. Conclusion： The optimizing method in constructing these recombinant plasmids serves other plasmid-based RNA interference research. The final plasmids PPARγ-pSUPER-EGFP established the basis for research on the function of PPARγ gene.     

Differentiation of NIH3T3 fibroblasts into adipocytes induced by peroxisome proliferator activated receptor γ2 expression

Objective To express mouse peroxisome proliferator activated receptor γ2 (mPPARγ2) in NIH3T3 fibroblasts mediated by the recombinant retrovirus and study its function. Methods The mPPARγ2 gene was subcloned into retrovirus vector pGCEN to generate the recombinant pGCEN/mPPARγ2. Then it was packaged into PA317 cells and selected with G418. Viral supernatants were harvested and then used to infect NIH3T3 fibroblasts. PPARγ activator 5,8,11,14-eicosatetraynoic acid (ETYA) was used to induce the mPPARγ2-expressing NIH3T3 cells into adipocyte differentiation.Results The recombinant retrovirus pGCEN/mPPARγ2 was constructed, and the higher titers of the viral supernatants were obtained. mPPARγ2 was expressed in NIH3T3 cells mediated by the recombinant retrovirus. Lipid accumulation obviously existed in these induced adipocytes which morphologically resembled mature adipocytes in vivo and expressed tissue specific adipocyte P2 (AP2) and Leptin genes.Conclusions An adipocyte differentiation model in vitro was successfully established. The work is the basis for further research on the molecular mechanism of adipocyte differentiation induced by PPARγ2.     

Purification and immunity analysis of recombinant 6His-HPT protein expressed in E. coli

Objective To obtain HPT protein (Hygromycin B Phosphotmnsferase), a kind of plant selective maker gene product expressed from E.coli and to prepare the polyclonal antibody (pAbs) against it. Methods HPT cDNA fragment was obtained by PCR and was inserted into the prokaryotic expressing vector pBV222. Then the constructed recombinant plasmid pBV222-HPT was transfered into E.coli DH5α for HPT expression. The recombinant expressing system was confirmed by restriction endonuclease digestion, DNA sequencing and protein expression. E.coli cells were lysed by sonication and detergent dissolution. After cell membrane was extracted, the inclusion bodies were denatured by 8 mol/L Urea and purified with metal chelate affinity chromatography on Ni-NTA agarose under denaturing condition. The purified 6His-HPT was characterized by SDS-PAGE, and used to immunize rabbit. The titer and specificity of antisera were detected by ELISA and Western blot respecitively. Results Analysis of DNA sequence and restricted enzymes showed that the sequence of PBV222-HPT plasmid was correct. The amount of recombinant HPT expressed in E.coli accounted for 30% of total cellular proteins. From 1 liter of fermentative bacteria about 22 milligrams of pure recombinant HPT was isolated with purity above 95%. The recombinant HPT protein could produce high titer antiserum in rabbits and show good immunity activity. Western blot showed specific binding reaction between the antiserum to the purified 6His-HPT protein and their expressed products (plants protein and bacterial protein). Conclusion HPT protein can be expressed and purified from E.coli by a relatively simple method, which has high immunity activity.     

Soluble expression of active human β-defensin-3 in Escherichia coli and its effects on the growth of host cells

Background Human β-defensin-3 （HBD3） is an epithelial peptide that has been demonstrated to have a salt-insensitive broad spectrum of potent antimicrobial activity. Expressing antimicrobial peptides in Escherichia coil （E. colt） is very difficult for it can result in death of the bacterial host cells. Our aim was to establish a prokaryotic system expressing soluble HBD3 protein and demonstrate the antimicrobial activity of the expressed protein. We then studied whether the host cells would activate the suicide pathways. Methods We first cloned the complementary DNA coding for the mature chain of HBD3, inserted it into the vector PGEX-KG then transformed E. coil BL21 （DE3） with the appropriate recombinant plasmid. After induction with 0.5 mmol/L isopropyl-1-thio-β-D-galactopyranoside （IPTG） the transformed E. cofiproduced a recombinant glutathione S-transferase and HBD3 （GST-HBD3） fusion protein. The fusion protein was treated with thrombin to produce pure HBD3 protein then the antimicrobial activity of HBD3 was evaluated in a liquid microdilution assay. Results The fusion protein GST-HBD3 was efficiently cleaved by thrombin and yielded HBD3 that had anti-staphylococcus aureus activity with a minimal inhibitory concentration level of 12.5 μg/ml. The E. coil strain expressing the recombinant protein did not grow slower than the empty vector strain. Conclusion Active HBD3 in E. coil by expressing the recombinant protein GST-HBD3 could be produced, and suicide did not occur in the E. coli strain expressing the recombinant protein.     

Construction, Expression and Identification of a Recombinant BCG Vaccine Encoding Human Mycobacterium Tuberculosis Heat Shock Protein 65

Heat shock protein 65 (HSP65) is one of the most important protective immunogens against the tuberculosis infection. The signal sequence of antigen 85B and the whole HSP65 DNA se-quence of human Mycobacterium tuberculosis (M. tuberculosis) were amplified from BCG genome and plasmid pCMV-MTHSP65 respectively by polymerase chain reactions (PCR). These two se-quences were cloned into the plasmid pBCG-2100 under the control of the promoter of heat shock protein 70 (HSP70) from human M. tuberculosis, yielding the prokaryotic shuttle expression plas-mid pBCG-SP-HSP65. Results of restriction endonuclease analysis, PCR detection and DNA se-quencing analysis showed that the two cloned DNA sequences were consistent with those previously reported, and the direction of their inserting into the recombinant was correct and the reading frame had been maintained. The recombinants were electroporated into BCG to construct the recombinant BCG vaccine and induced by heating. The induced expression detected by SDS-PAGE showed that the content of 65 kD protein expressed in recombinant BCG was 35.69% in total bacterial protein and 74.09% in the cell lysate supernatants, suggesting that the recombinant HSP65 gene could express in BCG with high efficiency and the expressed proteins were mainly soluble. Western-blot showed that the secretive recombinant proteins could specifically combine with antibody against M.tuberculosis HSP65, indicating that the recombinant proteins possess the biological activity of HSP65.     

Cloning and expression of the ureC geneo f H.pylori

AIM: To express Hp ureC gene in E.coli, a fragment from Hp ureC was amplified by the polymerase chain reaction and was cloned into E.coli expressing plasmid vector--- pBV220. This study lay the foundation for further studies of its functions. METHODS : An 1173bp gene fragment was amplified by the polymerase chain reaction. The template DNA was purified from Hp clinical strain. The gene fragment was digested by BamH I and EcoR I and cloned into pGEM-3Zf (-) plasmid. Its sequence was determined by autosequencing instrument from two directions. Then the gene fragment was cloned into pBV220. Subsequently the DH5 a was transformed by pBV220/ureC and was induced by heat when temperature reached 42“C. The form of expressed protein was analyzed to learn that in which form the recombinant protein was expressed. The thin layer chromatogram was used to scan thegel to learn that the quantity of the expressed protein. Antigenicity of the expressed protein wasconfirmed by ELISA and DIGFA. RESULTS z 95% of the DNA sequence we got is the same as that ofHp strain M60398. A Mr 44000 recombinant protein was induced by heat when temperature reached 42℃. The molecular mass of the expressed ureC gene is coincide with expected. The expressed proteinaccounts for 24.8% of the whole protein of the host bactria. Although most of the recombinant proteinsare inclusion bodies in host bactria after translation, there does exist certain quantity of solvable proteins in the supernatant of bacteria lysate. CONCLUSION：Recombinant H.pylori urease C protein is expressed in E.coli and the expressed protein has the antigenicity ofureC. Our study lays the foundation for further studies of the functions of lip ureC.     

Expression and purification of recombinant human hemangiopoietin in Escherichia coli

Objective： To express the soluble recombinant hemangiopoietin protein in E. coli BL21 （DE3）. Methods： Using human fetal live cDNA as a template, a partial cDNA fragment of HAPO coding N-terminal region was subcloned into plasmids pTrc99, pQE60 and pET32c to construct different recombinant prokaryotic expression systems. After selecting, the soluble rhHAPO fusion protein was expressed stably in E. coli BL21 （DE3） by vector pET32c-HAPO and further isolated by nickelnitrilotriacetic acid （NTA） affinity chromatography. After cleavage with enterokinase, the rhHAPO protein was applied to Fast Flow SP sepharose column. Results： The rhHAPO protein had a purity of more than 95% and a good bioactivity based on the cell adhesion assay in ECV304 cells. Conclusion： We have established a protein engineering system to produce rhHAPO which may provide the possibility for clinical application     

Cloning and expression in Escherichia coli of a new gene of Schistosoma japonicum encoding casein kinase Ⅱ beta subunit

Background Nowadays it is now a focus topic in schistosomiasis research to find ideal vaccinecandidates and new drug targets for developing anti-schistosomiasis vaccine. We cloned a new gene, casein kinase Ⅱ beta subunit, of Schistosoma japonicum (S. japonicum) and express it in Escherichia coli (E. coli). Methods The ESTs obtained in our laboratory were analyzed by homologous searching, and a new gene was recognized. The full-length cDNA of the new gene was obtained by joining the 3‘ RACE PCR fragment and the EST clone. To express the new gene, the cDNA was cloned into pGEX-4T-1 vector and then transformed into E. coli JM109. The recombinant protein was analyzed by SDS-PAGE and Western-blot. Results A 908 bp cDNA was isolated from S. japonicum and identified to be casein kinase Ⅱ beta subunit gene by sequence analysis. The open reading frame of the gene encodes a protein of 217 amino acids exhibiting 75.8%, 75.8%, 73.9%, 68.2%, 51.6% identity to the amino acids sequence of the corresponding genes of Homo sapiens (H. sapiens), Xenopus laevi (X. laevi) , Drosophila melanogaster (D.melanogaster), Caenorhabditis elegan (C. elegan), and Schizosaccharomyces pombe (S. promber) respectively. The predicted molecular weight of the protein was 24. 921 kDa. The new cDNA sequence had been submitted to GenBank, and its accession number is AY241391. This cDNA was subcloned into the pGEX-4T-1 vector and expressed in E coil JM109. The recombinant protein could be recognized by the S. japonicum infected rabbit serum. Conclusion The full-length cDNA sequences encoding S. japonicum casein kinase Ⅱ beta subunitwere firstly sequenced, cloned, and expressed in E coil.     

Promotion of chondrogenesis of marrow stromal stem cells by TGF-β3 fusion protein in vitro

The purpose of this study was to investigate the repair of the osteoarthritis（OA）-induced car- tilage injury by transfecting the new TGF-β3 fusion protein （LAP-MMP-mTGF-β3） with targeted ther- apy function into the bone marrow-derived mesenchymal stem cells （MSCs） in rats. The recombinant of plRES-EGFP-MMP was constructed by combination of DNA encoding MMP enzyme cutting site and eukaryotic expression vector plRES-EGFP. LAP and mTGF-β3 fragments were obtained from rat em- bryos by RT-PCR and inserted into the upstream and downstream of MMP from plRES-EGFP-MMP respectively, so as to construct the recombinant plasmid ofplRES-EGFP-LAP-MMP-mTGF-β3, plRES- EGFP-LAP-MMP-mTGF-β3 was transfected into rat MSCs. The genetically modified MSCs were cul- tured in medium with MMP-1 or not. The transfected MSCs were transplanted in the rat OA models. The OA animal models were surgically induced by anterior cruciate ligament transaction （ACLT）. The pathological changes were observed under a microscope by HE staining, Alcian blue, Safranin-fast Gre- en and graded by Mankin＇s scale, plRES-EGFP-LAP-MMP-mTGF-β3 was successfully constructed by means of enzyme cutting and sequencing, and the mTGF-β3 fusion protein （39 kD） was certified by Western blotting. Those genetically modified MSCs could differentiate into chondrocytes induced by MMP and secrete the relevant-matrix. The transfected MSCs could promote chondrogenesis and matrix production in rat OA models in vivo. It was concluded that a new fusion protein LAP-MMP-mTGF-β3 was constructed successfully by gene engineering, and could be used to repair the OA-induced cartilage injury.     

Induction of chondrogenesis of adipose-derived stem cells by novel recombinant TGF-β3 fusion protein

A new type of TGF-β3 fusion protein with targeted therapy function was constructed,and its feasibility and target specificity of inducing chondrogenesis were investigated by transfecting LAP-MMP-mTGF-β3 gene into adipose-derived stem cells (ADSCs).The recombinant pIRESEGFP-MMP was constructed by inserting the sense and antisense DNA of encoding the amino acid of the synthetic MMP enzyme cutting site into the eukaryotic expression vector pIRES-EGFP.LAP and mTGF-β3 fragments were obtained by using RT-PCR and inserted into the upstream and downstream of MMP from pIRES-EGFP-MMP respectively,and the recombinant plasmid of pIRES-EGFPLAP-MMP-mTGF-β3 was constructed,which was transferred to ADSCs.The ADSCs were cultured and divided in three groups:experimental group (MMP group),negative control group (no MMP) and non-transfection group.The morphological changes were observed microscopically,and the expression of proteoglycan and type Ⅱ collagen (ColⅡ) was detected by using Alcian blue staining and immunohistochemistry staining at 7th,14th and 21st day after culture.The recombinant plasmid of pIRES-EGFP-LAP-MMP-mTGF-β3 was correctly constructed by methods of enzyme cutting and sequencing analysis.The mTGF-β3 fusion protein was successfully expressed after transfection,and in the presence of the MMP,active protein mTGF-β3 was generated,which significantly promoted differentiation of ADSCs into chondrocytes and the expression of cartilage matrix.The novel fusion protein LAP-MMP-mTGF-β3 can targetedly induce differentiation of ADSCs into chondrocytes,which would open up prospects for target therapy of cartilage damage repair in future.