Cloning of hCD154 gene from human activated peripheral blood mononuclear cell and expression of hCD154-GST fusion protein in prokaryote

AIM:To express recombinant hCD154-GST fusion protein, to prepare anti-hCD154 monoclonal antibody, and to investigate the effect of anti-hCD154 monoclonal antibody on graft rejection. METHODS AND RESULTS: Total RNA was prepared from human peripheral blood mononuclear cell (PBMC) activated with 10ng/mL PMA and 1 μg/mL PHA for 8h, the total RNA was reversetranscribed to cDNA. The entire coding region and a part of the 3'non-coding regions were amplified by PCR using a pair of primers designed and synthesized according to the sequence of human CD154 gene from gene bank. The amplified product, a 820bp DNA fragment was cloned into pGEX-4T-1 plasmid expressing glutathione S-transferase(GST). The cloned insert was identified by double digestion of the cloned pGEX-4T-1 plasmid with retriction enzymes BamHⅠand EcoRⅠ.The fusion protein expression plasmid of PGEX-4T-1/hCD154 was constructed, then transformed to E coli BL21. The human CD154-GST fusion protein expression was induced by IPTG in BL21. The expression of recombinant 26kD GST and 55kD human CD154-GST fusion protein were confirmed by SDS-PAGE. CONCLUSION: We have express the recombinant human CD154-GST fusion protein. The expressed hCD154-GST fusion protein will be used to prepare anti-hCD154 monoclonal antibody, to investigate the role of anti-CD154 monoclonal antibody on graft rejection.     

Prokaryotic expression and preparation of GST/BFRF3 fusion protein of Epstein-Barr virus for screening nasopharyngeal carcinomas

AIM: To construct a prokaryotic expression plasmid containing Epstein-Barr viral (EBV) capsid antigen BFRF3 gene and to observe the application of recombinant BFRF3 protein in the serological diagnosis of nasopharyngeal carcinoma (NPC).METHODS: DNA extracted from the B95-8 cells was used as the templates. Polymerase chain reaction (PCR) was used to generate a DNA fragment of BFRF3 gene, and a 531-bp DNA fragment was inserted into a PGEX-5X-1 vector. The recombinant plasmid was transformed into E.coli BL21 (DE3). The expression of GST/BFRF3 fusion protein was induced by IPTG, identified by both SDS-PAGE and Western blotting, and then purified by glutathione-sepharose beads. The purified recombinant protein was coated to microplate for ELISA detection of EBV-IgA antibody in NPC patients.RESULTS: The GST/BFRF3 fusion protein was successfully expressed in E. coli. The molecular weight of the product was approximately 44 kD. The recombinant fusion protein GST/BFRF3 showed good immunoreactivity. A novel ELISA was established using GST/BFRF3 protein. Serum samples collected from the NPC patients and healthy controls were tested by this ELISA. The sensitivity and specificity of GST/BFRF3 tests for NPC patients were 65％ and 87％, respectively.CONCLUSION: The recombinant protein GST/BFRF3 is expressed in E.coli, and it has diagnostic value for screening of NPC patients.     

Effect of hemolysin protein HlyX of Leptospira interrogans serovar Lai on permeability of vascular endothelial cells

AIM: To clone and express the hemolysin gene hlyX of Leptospira interrogans serovar Lai and to investigate the effect of the expression product on the permeability of human umbilical vein endothelial cells (HUVECs).METHODS: The recombinant plasmid pET-hlyX was constructed by inserting the hlyX gene into prokaryotic expression vector pET32a(+), and transformed into E.coli BL21(DH3) to express the fusion protein Trx-HlyX with a His-tag.The fusion protein was purified using HisTrap affinity columns.The permeability of the monolayer HUVECs was measured by enzyme-linked immunosorbent assay for biotin-labeled albumin.Flow cytometry and Hoechst 33258 staining were applied to measure the apoptotic rate of HUVECs after incubation with Trx-HlyX.RESULTS: The recombinant plasmid pET-HlyX was successfully constructed and the fusion protein Trx-HlyX was highly expressed.Compared with the control cells, the purified recombinant protein Trx-HlyX significantly increased the permeability of transfected cells and promoted apoptosis of HUVECs (P<0.05).CONCLUSION: The recombinant plasmid pET-hlyX highly expresses the fusion protein Trx-HlyX.Purified protein Trx-HlyX influences the permeability and has cytotoxicity on HUVECs.     

Cloning expression and toxic effect on the host bacterial viability of an outer envelope protein from the strong virulent leptospira lai in China

AIM: Construction of an eukaryote-E. coli shuttle expressing recombinant plasmid which expresses OmpL1 envelope protein of pathogenic Leptospira, serovar Lai strain 017. METHODS: The OmpL 1 gene was amplified by PCR from the leptospiral genome. Then it was cut with restriction enzymes and ligated to the plasmid pBK-CMV. The correct recombinant plasmid was screened out with analysis of restriction enzymes and PCR. After inducing the E. coli baring recombinant plasmid with IPTG,the complete protein of the bacteria was extracted for SDS-PAGE. At the same time, OD600 of the host bacteria was examined at different time after inducing or uninducing with IPTG. RESULTS: Five strains E. coli containing proper recombinant plasmids were screened out. Four strains E. coli expressed a new protein with a weight of 37 kD among them. With the expression of the heterogenous protein,the OD600 of the host bacteria decreased. CONCLUSION: The shuttle expressing plasmid of the OmpL 1 gene of strong virulent Leptospira strain 017 was successfully constructed. Furthermore,the recombinant plasmid expressed the expected OmpL 1 fusion protein in E. coli and the expression of the heterogenous protein had toxic effect on the host bacteria. This work was important for the future research of OmpL1 protein which relates to the diagnosis,new vaccine preparing and the pathogenic mechanism of leptospirosis.     

欢迎订阅2006年下列期刊

AIM: To construct prokaryotic expression vector of His-tagged human IP-10 for further study of its biological function in the inflammatory response. METHODS: The coding sequence of IP-10 lacking signal peptide was amplified from human lung cDNA library by polymerase chain reaction (PCR) and the fragment was cloned into pET-14b plasmid for the construction of His-tagged fusion protein expressing vector, pET-14b/IP-10. After being identified by enzyme digestion and sequencing, the recombinant vector was transformed into a strain of E. coli, BL21 (DE3). The expression of His-tagged fusion protein was induced with IPTG and purified with Ni+-NTA affinity chromatography. Then the chemotactic activity of IP-10 was determined by transwell migration assay on THP-1 cells. RESULTS: The construction of pET-14b/IP-10 recombinant vector was proved by enzyme digestion and sequencing. The fusion protein IP-10, which was purified by a routine Ni+ affinity method, had an activity on the induction of cell migration of THP-1. CONCLUSION: We successfully construct IP-10 fusion protein expressing vector and get the fusion protein with high bioactivity, which provides essential materials for the future studies on IP-10. V     

辣椒脉斑驳病毒CP基因的原核表达及其抗血清的制备

采用RT-PCR方法克隆了辣椒脉斑驳病毒文昌分离物（ChiVMV-WC）的CP基因,并将其连接到原核表达载体pET-30b（+）上,克隆测序以确定其阅读编码框的正确性,然后将获得的重组质粒pET30b-ChiVMV CP转化大肠杆菌Rosetta（DE3）后,用IPTG进行诱导表达。SDS-PAGE分析结果表明,CP基因在大肠杆菌中获得了高效表达,获得的融合蛋白分子量约为38 kD。用Ni2+-NTA 琼脂糖亲和层析纯化的融合蛋白免疫兔子并获得抗血清。Western blot检测结果表明,抗血清与诱导表达的ChiVMV-WC编码CP蛋白发生特异性反应。间接酶联免疫吸附法（ID-ELISA）检测抗血清效价为1/106。通过对田间20个样品的ID-ELISA检测,证实了所制备的抗血清与ChiVMV病叶具有良好的反应特异性。     

‘三棱榄''橄榄果实香气成分分析

1 材料与方法选取广东优良鲜食橄榄品种‘三棱榄’,2001年12月6日采样,采用固相微萃取法(SPME)富集香气成分(鲜橄榄果肉于15℃下捣碎后取样1.0 g放入4 mL聚四氟乙烯硅橡胶垫密封螺口玻璃瓶中,插入100μm聚二甲基硅氧烷纤维头于室温25-30℃顶空取样2 h),用美国Finnigan TRACE GC-MS气相色谱-质谱联用仪进行分析。气相色谱柱为DB-1弹性毛细管柱30 m×0.25 mm,载气为He(99.99％),流速1.0mL/min。程序升温从40℃开始先保持10 min,后以2℃/min的升温速率升至150℃保持10 min。质谱条件:电子能量70 eV,离子源温度250℃,质量范围35-450 aum,不分流进样。2002年12月18日采样重复分析。     

Soluble expression of a CXCL10-loop3-EGF fusion protein and its anti-tumor activity

AIM: To evaluate the implication of CXCL10-loop3-EGF fusion protein for the activities of targeting tumor and anti-angiopoiesis. METHODS: RT-PCR was preformed to amplify CXCL10 coding sequence from PBMC activated by IFN-γ. CXCL10-loop3-EGF fusion gene, which was conducted by Over-Lap Extention PCR, was hinged up with plasmid pTG19-T, transfected to E. coli DH5α and processed positive colony selection. After ligated with plasmid pET32a(+), recombinant CXCL10-loop3-EGF fusion gene was then transfected to E. coli Origami B (DE3) and induced to express its coding fusion protein his-CXCL10-loop3-EGF. The recombinant fusion protein CXCL10-loop3 -EGF was purified by His-bind affinity chromatograph, enterokinase cleavage, ultrafiltration and dislysis. The transwell chemotatic test and HUVEC angiopoiesis inhibition test were performed to determine the anti-tumor responses and anti-angiopoiesis activity of CXCL10-loop3-EGF fusion protein. RESULTS: CXCL10-loop3-EGF fusion protein was successfully constructed and confirmed by SDS-PAGE analysis and Western blotting. Significant PBMC chematatic activity and HUVEC anti-angiopoiesis activity were observed. CONCLUSION: CXCL10-loop3-EGF fusion protein, which has perfect anti-tumor activity, is successfully constructed.     

Expression of fusion protein anti-HER2-ScFv-GFP in insect cells and binding to surface of breast cancer cells

AIM: To express a recombinant fusion protein anti-human epidermal growth factor receptor-2 single-chain variable fragment with green fluorescent protein (anti-HER2-ScFv-GFP) using the insect cells-Bac-to-Bac baculovirus expression system and to analyze the binding function of this fusion protein with HER2 on the surface of the breast cancer cells. METHODS: Human anti-HER2-ScFv gene from mice was fused with GFP gene. To obtain the recombinant plasmid pFAST Bac-to-Bac HT A/anti-HER2-ScFv-GFP, we inserted it into Bac-to-Bac baculovirus expression plasmid pFAST Bac-to-Bac HT A. The identified recombinant plasmid was transferred into Escherichia coli DH10Bac to allow the generation of a recombinant bacmid. After transfected the recombinant virus bacmid into the insect cells Tn-5B1-4, the recombinant virus was collected to infect Tn-5B1-4. SDS-PAGE and Western blotting analysis were used to verify the expression product in Tn-5B1-4. The fusion protein was purified with Ni²⁺-NTA affinity chromatography. The purified fusion protein was bound to the surface of HER2-positive breast cancer cells SKBR3 and HER2-negative breast cancer cells MCF7. The binding effects on the surface of breast cancer cells were observed under laser confocal microscope. RESULTS: The fusion gene anti-HER2-ScFv-GFP was successfully constructed with the length of 1 539 bp. The green fluorescence was also observed in Tn-5B1-4 cells infected with the recombinant virus under fluorescent microscope. A 60 kD protein was examined and confirmed by SDS-PAGE and Western blotting. Under laser confocal microscope, strong green fluorescence was observed on the surface of the HER2-positive breast cancer cells SKBR3. However, no green fluorescence was observed on the surface of HER2-negative breast cancer cell MCF7. Obvious green fluorescence on the surface of HER2-positive breast cancer cell SKBR3 was also found after the cells were eluted with 1×PBS. CONCLUSION: The fusion protein anti-HER2-ScFv-GFP was successfully expressed in insect cells Tn-5B1-4, and can firmly bind to the surface of breast cancer cells SKBR3 and emit the green fluorescent light.     

罗汉果葡萄糖基转移酶基因的克隆及原核表达

 从罗汉果（Siraitia grosvenorii）转录组中获得一条与罗汉果甜苷Ⅴ生物合成相关的葡萄糖基转移酶（UDPG）的unigene片段,以罗汉果授粉后70 d的果实RNA为模板,利用RACE和RT-PCR技术克隆UDPG全长基因,将克隆得到的SgUDPG1基因连接到原核表达载体pEASY-E1上,构建融合表达载体,转化到大肠杆菌BL21（DE3）,通过IPTG诱导表达,重组蛋白纯化,SDS-PAGE检测表达产物以及Western-blotting和质谱鉴定蛋白产物。结果表明,获得了1条SgUDPG1,全长为1 959 bp,开放阅读框ORF为1 365 bp,编码1条454 aa的肽链,理论分子量为51.2 kD,等电点为5.39,具有植物中次生代谢产物糖基转移酶特有的保守结构域PSPG-box motif。SgUDPG1在授粉后50 d和70 d的果实中表达逐渐升高,是对照授粉后3 d的5.16倍和13.12倍,与果实中甜苷Ⅴ含量呈相同趋势。此基因的ORF可以在大肠杆菌中表达,并且可以纯化出比理论分子量大5.3 kD的融合蛋白,通过Western-blotting和质谱鉴定,确定该蛋白属于罗汉果葡萄糖基转移酶。     

Prokaryotic expression of Staphylococcus aureus Panton-Valentine leukocidin and its cytolytic activities to human polymorphonuclear neutrophils

AIM:Panton-Valentine leukocidin (PVL) is a pore-forming toxin secreted by Staphylococcus aureus epidemiologically associated with the often-lethal necrotizing pneumonia. Until now, the mechanisms of pathogenesis of PVL leading to the fatal pulmonia remains undefined and also acquired plenty of the toxins is difficult. In the present study, we obtain recombinant staphylococcal F and S components of the Panton-Valentine leukocidin by gene engineering and evaluate its biological activity in vitro, which provides an experimental basis for the further studies of its biological function and its toxicity in pneumonia. METHODS:The full-length of F and S components of PVL gene amplified from the strain of Staphylococcus aureus DNA by high-fidelity PCR was cloned into prokaryotic expression vector pET22b(+), and the vector was transformed into BL21 (DE3)plysS to construct a prokaryotic expression system. The integrity of the opening-reading frame of each construct was verified by DNA sequencing. The recombinant PVL (rPVL) was induced by1.0 mmol/L IPTG. The expressed products were identified by SDS-PAGE and the fusion proteins (6His-LukS-PV and 6His-LukF-PV) were purified from lysates of transfected E. coli cells by affinity chromatography on nitrilotriacetic acid columns. The cytolytic activity was tested by incubation of rPVL with human polymorphonuclear neutrophils (PMNs) in vitro. RESULTS:The nucleotide sequence of the cloned PVL gene was the same as that of reported in GenBank. E. coli BL21 (DE3)plysS containing recombinant vectors grow at 37℃ causes some proteins to accumulate as inclusion bodies, while incubation at 30 ℃ led to a significant amount of soluble active proteins which accounted for about 31.7% of the total bacterial protein.The relative molecular weight showed on SDS-PAGE profile was consistent with the expected value which the LukS-PV protein was about 34 kD, and the LukF-PV protein was about 35 kD. The purified rPVL was obtained and its cytolytic activity to PMNs was demonstrated. CONCLUSION:The genes of lukS-PV and lukF-PV are successfully cloned into plasmid pET22b(+) and expressed in E. coli respectively, which provide a basis for analyzing the toxicity related to the diseases and further studies about the pathogenesis of PVL.     

Construction of prokaryotic expression vector of human angiogenesis inhibitor arresten and its expression in E.coli

AIM:To construct prokaryotic expression vector of human angiogenesis inhibitor arresten gene and express recombinant arresten in Escherichia coli.METHODS:Human arresten gene was amplified from recombinant plasmid pGEM-Arr with polymerase chain reaction (PCR), and then cloned into prokaryotic expression vector pRSET by means of recombinant gene technology. The recombinant plasmid pRSET-Arr was transformed into E.coli BL21(DE3), and recombinant arresten was expressed in the bacteria under induction of IPTG. The expressed products were detected by SDS-PAGE analysis.RESULTS:Restriction analysis indicated that the arresten gene was successfully inserted into the expression vector, and DNA sequencing verified that the reading frame of the recombinant vector was correct. Recombinant arresten was successfully expressed in Escherichia coli; its molecular weight was about 26 kD and its amount was approximately 30% of total bacterial proteins.CONCLUSION:The successful construction of prokaryotic expression vector containing human arresten gene and the effective expression of recombinant arresten in Escherichia coli laid the foundation for further study on its biological functions.     

Construction of the vector for fusion protein gene driven by IGF-Ⅱ P3 promoter and its expression in hepatocellular carcinoma cells

AIM:To construct the shuttle plasmid vector for thymidine kinase(tk) and EGFP fusion protein gene driven by IGF-Ⅱ P3 promoter,and investigate the specific killing effect of the HSV-tk/GCV system on hepatocellular carcinoma(HCC) cells in vitro.METHODS:Recombinant shuttle plasmid vector was constructed by techniques of genetic recombination and screening,and identified by restriction digestion and sequencing analysis. Then the recombinant shuttle plasmid was transfected into HepG2 and HeLa cells by techniques of lipofectamine transfection and its expression was detected by fluorescence microscope and RT-PCR. Cell killing after ganciclovir(GCV) application was determined by MTT.RESULTS:Identification of pDC316-tkEGFP-P3 by enzyme digestion and sequencing analysis showed that the length,inserted location and direction of the target genes which were inserted into the recombinant were correct. It was found that enhanced green fluorescence protein could only be seen in HepG2 cells,but not in HeLa cells. The results of RT-PCR showed that only two bands could be seen in the samples of pDC316-tkEGFP-P3 transfected HepG2 cells. The MTT test showed the selective cytotoxicity of GCV to the transfected HepG2 cells.CONCLUSION:The shuttle plasmid vector carrying the tkEGFP fusion protein gene driven by IGF-Ⅱ P3 promoter has been constructed successfully and its specific expression in HepG2 cells provided a sound basis for targeted gene therapy for HCC.     

甘蔗黄叶病毒P0基因的克隆与原核表达及抗血清制备

根据已发表ScYLV-P0基因系列设计特异性引物,应用RT-PCR技术从甘蔗病叶的mRNA扩增得到目的DNA片段.以pET32a（＋）为原核表达载体,构建重组表达质粒pET32a-P0.经过双酶切鉴定和DNA测序后,将重组表达质粒转入大肠杆菌BL21 （DE3）pLySs,在30℃培养条件下IPTG诱导表达.通过SDS-PAGE电泳检测融合蛋白表达情况.表达结果显示,在该表达系统中,融合表达蛋白P0是以包涵体形式的蛋白存在;P0融合蛋白大小约45kDa,与P0开放阅读框的理论推算值29.991 kDa加上载体自身蛋白约18 kDa相符,用Ni2＋-NTA琼脂糖亲和层析纯化融合蛋白,免疫家兔制备出抗血清,通过酶联法（ID-ELISA）测定本实验制备的ScYLY-P0抗血清工作浓度为1：25000.     

Construction of pNTAP-PRAK eukaryotic expression plasmid and its expression in HEK293 cells

AIM: To construct pNTAP-PRAK eukaryotic expression plasmid and to establish a stable HEK293 cell line expressing tandam affinity purification (TAP)-tagged PRAK. METHODS: Human PRAK coding region was subcloned into pNTAP vector to construct a recombinant plasmid called pNTAP-PRAK, then DH5α E.coli was transformed with the recombinant plasmid. After identified by PCR, digestion with restriction endonuclease and sequencing, the correct recombinant expression plasmid was transfected with PolyFect liposome transfection reagent to HEK293 cells. The cell line with stable expression of exogenous TAP tagged-PRAK gene was established by screening of antibiotic G418. The expression and localization of the fusion protein TAP tagged-PRAK were detected by Western blotting and immunofluorescence assay. RESULTS: All the results of identification by PCR, digestion with restriction endonuclease and sequencing indicated that the recombinant eukaryotic expression plasmid pNTAP-PRAK was constructed correctly. The result of Western blotting showed that the recombinant plasmid was expressed stably in HEK293 cells after transfection followed by G418 screening. The result of immunofluorescence assay showed that the expression product TAP tagged-PRAK distributed mainly in the nucleus. CONCLUSION: The eukaryotic expression vector pNTAP-PRAK was successfully constructed and the cell line stably expressing TAP tagged-PRAK was established. TAP tag didnt influence the localization of exogenous PRAK.     

Expression and purification of re-constructed human CRP and observation of its internalization into HeLa cells

AIM: To construct prokaryotic expression vector for human C-reactive protein (CRP), to acquire the functional fusion protein purified from BL21(DE3) transformed with vector pET14b/EGFP-hCRP, and to observe the internalization of the fusion protein his-EGFP-CRP into tumor cell line HeLa. METHODS: CRP gene sequence was amplified with the vector p91023/CRP as template by PCR, and was inserted into vector pET14b/MCS-EGFP-(N)36 to construct prokaryotic expression vector. The E. coli cells BL21(DE3) transformed with the re-constructed vector pET14b/EGFP-hCRP was induced by isopropyl-β-D-thiogalactopyranoside (IPTG), and the expressed protein his-EGFP-CRP were purified with affinity chromatography method and refolded with gradient filtration. The HeLa cells were observed under the fluorescence microscopy after the addition of purified renature protein. RESULTS: The results of identification by PCR, digestion with restriction endonuclease and sequencing indicated the construction of vector pET14b/EGFP-hCRP was correct; the SDS-PAGE showed that the transformed E. coli cells could be induced to express the fusion protein his-EGFP-CRP and the purification of proteins were successful. We could found fluorescent signal around the cell membranes, in the cytoplasm and nuclei in the observation of the HeLa cells incubated with his-EGFP-CRP. CONCLUSION: The prokaryotic expression vector for human CRP linked with his and EGFP coding sequence is successfully constructed. The fusion protein his-EGFP-CRP is purified and refolded. The reconstructed protein expressed by prokaryotic cells adheres to the membrane of tumor cell HeLa and is internalized into the cytoplasm and nuclei of the cells.     

筇竹CBF1基因的原核表达和多克隆抗体的制备

以筇竹（Qiongzhuea tumidinoda Hsueh et Yi）叶片为试材,采用RT-PCR方法克隆其CBF1基因,并将CBF1连接到原核表达载体pET32a（+）上,经克隆测序确定所构建的重组载体pET32-QZ开放阅读框正确。将重组载体pET32-QZ转化大肠杆菌Rosetta2（DE3）菌株,经IPTG诱导表达,SDS-PAGE凝胶电泳,考马斯亮蓝染色,证明CBF1蛋白得到了高效表达,所表达蛋白是大小约为45 kD的融合蛋白。经镍柱纯化后作为抗原免疫家兔,制备CBF1蛋白特异性抗血清。所制备的多克隆抗体能够与融合蛋白和经冷诱导的筇竹叶片总蛋白在25 kD处出现杂交条带。上述结果表明,表达的目的蛋白可用于免疫组织化学、蛋白质印迹检测。     

Effect of adipose differentiation-related protein on proliferation and apoptosis in H9c2 cells

AIM: To construct an adipose differentiation-related protein (ADRP) eukaryotic expression vector and to explore the effect of ADRP on apoptosis of H9c2 cells induced by palmitic acid (PA). METHODS: The ADRP gene obtained by the method of RT-PCR was cloned into pEGFP-C1 plasmid. The recombinant plasmid was transformed into E.coli DH5α for amplification. The recombinant plasmid was extracted from E.coli DH5α and transfected into H9c2 cells by Lipofectamine^TM2000. The stable transformants were selected by G418 screening. Expression of green fluorescent protein was observed under fluorescence microscope and the ADRP expression was identified by RT-qPCR and Western blotting analysis. The effect of PA on the proliferation of H9c2 cells was detected by MTT assay. The apoptotic percentage of H9c2 cells caused by PA was determined by flow cytometry. RESULTS: The eukaryotic expression vector pEGFP-C1-ADRP was successfully constructed. Green fluorescent was observed in the cells transfected with pEGFP-C1 or pEGFP-C1-ADRP under fluorescence microscope. RT-qPCR and Western blotting analysis showed that recombinant cells exhibited high mRNA and protein levels of ADRP. After treated with PA at different concentrations, the apoptosis rates and the proliferation inhibition of recombinant cells were both lower than those of the other two cells. CONCLUSION: The transfected H9c2 cells with stable ADRP expression were successfully established. The over-expression of ADRP prevents the cells from apoptosis and inhibition of proliferation caused by PA, indicating that ADRP plays a protective role in H9c2 cells.