Construction of eukaryotic expression vector and expression of outer membrane lipoprotein LipL41 gene of Leptospiria lai

AIM:To construct a eukaryotic expression vector expressing outer membrane lipoprotein LipL41 of Leptospira lai and express it in mammalian cell. METHODS:LipL41 gene was amplified by PCR from genome of Leptospira lai 017 strain, and was subcloned into vector pGEX-4T-1. After sequencing, LipL41 gene digested by restriction endonuclease and cloned into vector pcDNA3. After confirming the correctness of the eukaryotic recombinant vector by restrication enzyme digestion, it was transfected into COS7 cells by liposome. Its expression was analyzed by RT-PCR. RESULTS:A fragment of 1 011 bp was amplified, and sequence analysis showed it had a 98% homology with Leptospira kirschneri. The analysis of restriction enzyme indicated that the eukaryotic recombinant vector was correctly constructed. A specific amplified fragment was showed in the cells transfected with recombinant plasmid by RT-PCR, but the cell transfected with blank plasmid did not show this band. CONCLUSIONS:The LipL41 gene of Leptospira lai was successfully inserted into eukaryotic expression plasmid and the recombinant plasmid expressed the LipL41 mRNA.     

Effect of estrogen receptor 2 on the cell proliferation in prostate cancer cell line PC-3M

AIM: To construct the recombination plasmid pcDNA3.1-hERβ with the human estrogen receptor 2 (ESR2) full length cDNA and transfect it into hormone-independent prostate cancer PC-3M cell line, and to study the effects of ESR2 on proliferation in transfected cells. METHODS: The complete cDNA of ESR2 was obtained from human ovary tissue by RT-PCR technique and cloned into eukaryotic expression vector pcDNA3.1 by using gene recombination technique to construct the pcDNA3.1-hERβ recombination plasmid. The plasmid was detected by endonuclease digestion and DNA sequencing and was transfected into PC-3M cells. MTT and FCAS assay were used to test the effects of ESR2 on the ability of proliferation in PC-3M cells. RT-PCR and Western blotting were used to detect the expressions of cyclinD1 and P21Cip1. RESULTS: The results of sequencing and endonuclease digestion demonstrated that the construction of pcDNA3.1-hERβ recombination plasmid was successful. The sequence analysis suggested that the ESR2 sequence detected by PCR was identical to that published in GenBank, and the product of endonuclease was as long as the complete human ESR2 gene. 48 h after transfected the pcDNA3.1-hERβ into PC-3M cells, the expression of ESR2 mRNA and protein levels increased significantly detected by RT-PCR and Western blotting. Compared to the cells transfected with vector as control, the PC-3M cells transfected with pcDNA3.1-hERβ showed that cell population decreased and proliferation activity degraded. FCAS showed that the cells in G0/G1 stage increased and in S stage or G2/M stage decreased. RT-PCR and Western blotting showed that the expression of cyclinD1 gene reduced and expression of P21Cip1 increased. CONCLUSION: The recombination of plasmid pcDNA3.1-hERβ is constructed and transfected into the PC-3M cells successfully. The activity of cell proliferation is inhibited after pcDNA3 transfection.1-hERβ. It is possible that ESR2 inhibits cell proliferation by the expression of proliferation related genes cyclinD1 and P21Cip1.     

Construction of mouse pdx-1 gene eukaryotic expression vector and its expression in embryonic stem cells

AIM: To clone mouse pdx-1 gene and construct its eukaryotic expression vector for expression of pdx-1 in mouse embryonic stem cells.METHODS: Mouse pdx-1 cDNA fragment was amplified with polymerase chain reaction (PCR) from mouse pancreatic cDNA. The purified fragment was recombinated with a eukaryotic expression vector carrying enhanced green fluorescent protein, pEGFP-N1. The pdx-1 cDNA fragment was inserted into the multi-clone sites of the vector to construct a new plasmid, pEGFP/pdx-1. E.colli strain DH5α was transfected with the new recombinant plasmid to expand it. Plasmid DNA extracted from the expanded DH5α was identifed by cutting with Hind Ⅲ, BamHⅠ nuclease and by DNA sequencing. Identified plasmid DNA was transfected into mouse embryonic stem cell line MESPU13 by carrying with liposome. RESULTS: A 876 bp cDNA fragment was amplified from mouse pancreatic cDNA by PCR and it was inserted into the vector pEGFP-N1 correctly. The fragment was defined to be pdx-1 gene by nuclease digestion and DNA sequencing. Mouse embryonic stem cell line MESPU13 was transfected with the new recombinant plasmid DNA. The green fluorescent protein report gene and pdx-1 gene expressed in transfected mouse embryonic stem cells within 24 h. CONCLUSION: Mouse pdx-1 gene is cloned and its recombinant eukaryotic expression vector carrying green fluorescent protein is constructed successfully. It provides a useful tool for further research on the function of pdx-1.     

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.     

Construction of eukaryotic expression vector of fusion gene CD80-IgG and its expression in Chinese hamster ovary cells

AIM: To construct the eukaryotic expression vector CD80-IgG by fusing the cDNA encoding extracellular portion of murine CD80 to the 5'-terminus of cDNA encoding Fc fragment of murine immunoglobulin G1 and to express the fusion protein in Chinese hamster ovary (CHO) cells. METHODS: The two cDNAs was amplified by PCR respectively from plasmid pcDNA/B7 containing the full-length cDNA of murine CD80 from murine spleen cells, and cloned to the eukaryotic expression vector pcDNA3.0 by directional cloning. The resultant recombinant plasmid pcDNA/CD80-IgG was transfected into CHO cells with liposome transfection reagent. The stably expressing cells were obtained by G418 screening. Western blot, Dot ELISA, and flow cytometry were used to detect the expression of the fusion protein and its immunological activity. RESULTS: DNA sequencing verified the correction of the construction of recombinant plasmid pcDNA/CD80-IgG. The expressed fusion protein was detected in the supernatant of transfected CHO cells and the molecular weight of the protein was similar to what we expected. Its immunological activity was also established. CONCLUSION: The recombinant plasmid pcDNA/CD80-IgG was successfully constructed and it expressed the fusion protein CD80-IgG.     

Macrophage migration inhibitory factor gene expression is suppressed by using antisense RNA mediated by expression vector

AIM:To study the potential of using antisense RNA mediated by expression vector to suppress MIF expression. METHODS:MIF gene was sub-cloned into plasmid pcDNA3 to construct MIF antisense RNA expression vector, pcDNA3-antiMIF, which was identified by restriction enzyme digestion and DNA sequencing. By using lipofectamine 2000, plasmid pcDNA3 and pcDNA3-antiMIF were transfected into MIF expression cells, 293-MIF, separately. 60 h later, the 293-MIF cells were collected and used to determine the MIF mRNA expression by real-time quantitive PCR. Plasmid pcDNA3-antiMIF was transformed into HUVECs, named HUVECs-antiMIF, to express MIF antisense RNA. HUVECs-antiMIF was screened by sulfate G418 and identified by PCR and RT-PCR analysis. Then the MIF expression vector, pSecTag-MIF, was transfected into HUVECs-antiMIF, and the MIF mRNA expression in HUVECs-antiMIF was determined by quantitative PCR. RESULTS:Restriction enzyme digestion and DNA sequencing analysis showed that MIF antisense RNA expression vector, pcDNA3-antiMIF, was constructed correctly. The results of quantitative PCR showed that MIF mRNA expression was suppressed by MIF antisense RNA at level about 32% (P<0.05) in 293-MIF cells. The HUVECs-antiMIF, which expressed MIF antisense RNA, was obtained and identified by PCR and RT-PCR assay. The results of quantitative PCR revealed that MIF mRNA expression was also down-regulated by about 40% (P<0.05) in HUVECs-antiMIF cells. CONCLUSION:MIF expression was suppressed efficiently by MIF antisense RNA mediated by expression vector, and the HUVECs-antiMIF was established to express MIF antisense RNA.     

Cloning and sequence analysis of death associated protein kinase gene ORF and DAPK1 inducing Raji cell apoptosis

AIM: Open reading frame(ORF) of death associated protein kinase1(DAPK1) gene was cloned for studying on tumor forming and metastasis.METHODS: Based on nucleotide sequence of DAPK1 gene from GenBank, a pair of primers was designed. DAPK1 gene ORF was transfected into Raji cells in expression vector pcDNA3.1(+) with lipofectamine reagent. Morphologic assessment of apoptosis was performed with fluorescence microscope cytotoxicity and cell viability was assayed by MTT. RESULTS: DAPK1 gene ORF was amprified from K562 cells by RT-PCR. It was cloned into plasmid pMD18-T and sequenced. There were seven mutation in 4 300 bp nucleotide sequence relatively to DAPK1 nucleotide sequence from GenBank, but six was synonymous mutation and one was single nucleotide polymorphism. 4 300 bp nucleotide of DAPK1 gene ORF was transfected into Raji cells. DAPK1 gene expression was detected in 48 h after it was transfected into Raji cells. Then Raji cells showed apoptosis. CONCLUSION: Large fragment gene was cloned by RT-PCR and transfected into Raji cells successfully. Over-expression of DAPK1 gene induced Raji cells apoptosis.     

Construction of Pax-8 gene recombinant plasmid and study of its function

AIM: To investigate the effects of over-expression of Pax-8 gene on the proliferation and apoptosis of H9c2 cells(a cardiomyocyte cell line). METHODS: The full length of rat Pax-8 gene was restrictively digested by Kpn I and Not I from the pCMV sport6-Pax-8 vector, and then inserted into the eukaryotic expression vector pcDNA3.1(+). The recombinant plasmid pcDNA3.1(+)-Pax-8 was confirmed by restriction endonuclease digestion and sequencing. The pcDNA3.1(+)-Pax-8 was transfected into H9c2 cells. The expression of Pax-8 at mRNA and protein levels was identified after transfection by RT-PCR and Western blotting. The cell proliferation was measured by CCK-8. Cell apoptosis was induced by serum deprivation in H9c2 cells transfected with Pax-8 gene. The apoptosis rate of the cells was determined by flow cytometry with annexin V-FITC and propidium iodide double staining. The protein expression of activated caspase-3 was measured by Western blotting. RESULTS: The full length of Pax-8 gene was successfully cloned into pcDNA3.1(+) expression vector and over-expression of Pax-8 at mRNA and protein levels was observed in H9c2 cells transfected with Pax-8 gene as compared to the wild-type cells and the cells transfected with an empty vector (both P<0.05). Transfection of Pax-8 gene promoted the proliferation of the cardiomyocytes (P<0.05) and inhibited the apoptosis rates induced by serum deprivation (P<0.01). The expression level of activated caspase-3 was increased by serum deprivation and attenuated by Pax-8 transfection (P<0.01). CONCLUSION: The pcDNA3.1(+)-Pax-8 expression vector was successfully constructed and over-expression of Pax-8 gene in cardiomyocytes is obtained. Pax-8 gene acts as an anti-apoptotic factor in cardiomyocytes by promoting cell proliferation and inhibiting apoptosis.     

Construction of an immunoconjugate targeting tissue factor and its effect on colorectal cancer cells

AIM: To construct a recombinant eukaryotic expression vector pcDNA3.1(+)-hFVII-LC+hIgG1-Fc, and to produce and purify the immunoconjugate hFVII-LC+hIgG1-Fc protein. METHODS: The target sequences were amplified by RT-PCR from hepatic tissue and lymphocyte RNA, and cloned into eukaryotic expression vector pcDNA3.1(+). After confirmed by restriction endonuclease digestion and DNA sequencing, the recombinant plasmid was transfected into CHO-K1 cells by lipofectamine 2000. The transfectant clones were selected by G418 screening. The positive monoclonals were grown in CHO-K1 serum-free medium Excel 301 and the culture medium was collected. The hFVII-LC+hIgG1-Fc protein was purified by affinity Ni-NTA resin. The immunoconjugate was identified by ELISA with tissue factor (TF) affinity and specificity. Induction of NK cell-mediated antibody-dependent cell cytotoxicity(ADCC) was examined in HT-29 colorectal cancer cell line. RESULTS: Human liver tissue and lymphocytes from Han population were used as template for amplification of hFVII-LC and hIgG1-Fc DNA fragments, which were confirmed by sequencing and were exactly the same as those GenBank reported. The eukaryotic expression vector pcDNA3.1(+)-hFVII-LC+hIgG1-Fc was successfully constructed, and 1.3 mg of hFVII-LC+hIgG1-Fc protein could be prepared from 1 liter of Excel 301 serum-free culture medium through Ni-NTA affinity chromatography. The immunoconjugate was specially bound to TF and induced a significant ADCC response in HT-29 cells. CONCLUSION: The human hFVII-LC+hIgG1-Fc recombinant plasmid and the hFVII-LC+hIgG1-Fc immunoconjugate are obtained, which provide the basis for further study of cancer-targeted therapy.     

Effect of recombinant connective tissue growth factor on the phenotype transition of rat adventitia fibroblasts

AIM:To construct pcDNA3.1(+)/connective tissue growth factor (CTGF) eukaryotic expression plasmid and to investigate its role in the promotion of phenotypic transition in adventitia fibroblasts (AF). METHODS:The expression vector pcDNA3.1(+)/CTGF was constructed by routine molecular biological method. The expression vector pcDNA3.1(+)/CTGF was confirmed by restriction enzyme digestion and sequencing method. The expression vector pcDNA3.1(+)/CTGF was transfected into AF and the exogenous expression was observed. The expression of the α-SM 〖JP+1〗actin was examined by Western blotting. RESULTS:The eukaryotic expression vector of CTGF was successfully constructed, which was transfected into AF, the expressed CTGF promoted phenotype transition in AF. CONCLUSION:The pcDNA3.1(+)/CTGF plasmid was constructed and transfected into AF, the expressed CTGF promoted phenotype transition in AF.     

Effect of human scavenger receptor SR-A I overexpression on uptaking ox-LDL and adhesion in 293T cells

AIM: To construct the recombinant eukaryotic expression plasmid pEGFP-C1-SR-A I for the high expression in 293T cells in order to identify functions of savenger receptor-A I (SR-A). METHODS: The primer was designed according to MSR1 cDNA and pEGFP-C1-SR-A I was constructed by standard molecular cloning technique and enzyme digestion. After sequencing, the plasmid was transfected into 293T cells by lipidosome method. The expression of scavenger receptor-A I was identified by RT-PCR and Western blotting. The foam cells were evaluated by the formation of lipid granules in the cells with oil red staining. Cell adhesion was analyzed by cell adhesion assay. RESULTS: 24 h after transfection, SR-A I mRNA was highly expressed and the high level of the protein was detected. The ratio of foam cell formation was doubled, the efficacy of cell adhesion was enhanced two times compared to the control group and the empty vector group. CONCLUSION: The recombinant eukaryotic expression plasmid has been constructed successfully with enhancing the function of uptake ox-LDL and adhesion in 293T cells by overexpression of SR-A I.     

Cloning and expression of NK4 gene in Raji cells

AIM: To clone NK4 gene and to construct recombinant eukaryotic expression vector for observing its expression in transfected Raji cells. METHODS: Total RNA was extracted from human hepatic tissue. NK4 gene cDNA was amplified by RT-PCR, and then cloned into vector pVITRO2-mcs to construct the recombinant eukaryotic expression vector pVITRO2-mcs-NK4. Raji cells were transfected by recombinant vector pVITRO2-mcs-NK4 and screened by homomycin B. The stable strain of NK4 gene expression was screened by real-time fluorescent quantitative PCR, ELISA, immunocytohistochemistry and semisolid culture. RESULTS: The specific DNA fragment was detected by RT-PCR in Raji cells transfected with NK4 gene. The transfected Raji cells expressed NK4 mRNA and protein stably, which inhibited Raji cell proliferation, metastasis and invasion. CONCLUSION: NK4 gene is cloned and recombined to construct recombinant eukaryotic expression vector pVITRO2-mcs-NK4 successfully. NK4 gene in Raji cells expresses stably.     

Effect of antisense RNA targeting Polo-like kinase 1 on cell growth in A549 lung cancer cells

AIM: To investigate the effect of Polo-like kinase-1 (Plk1) depletion on cell cycle progression and cell growth in lung cancer cells.METHODS: A recombinant plasmid containing antisense RNA targeting Plk1 (pcDNA3-Plk1) was transfected into A549 cells by lipofectine. RT-PCR and Western blotting were used to examine Plk1 gene expression. Cell proliferation was evaluated by cell counting and BrdU labeling. Cell cycle distribution and apoptosis were examined by flow cytometry. Inhibition rate (IR) of vinorebline (NVB) was determined by MTT assay. RESULTS: After transfected with pcDNA3-Plk1 into A549 cells, the expression levels of Plk1 mRNA and protein were greatly decreased. Abnormal morphological changes of cells and growth inhibition were observed in pcDNA3-Plk1 transfected cells. The BrdU labeling index was significantly lower than that in control group (P<0.05). Cells showed a strong G₂/M arrest and apoptosis 72 h post transfection. IR of vinorebline in pcDNA3-Plk1 transfected groups was significantly higher than that in other groups. CONCLUSION: Antisense RNA targeting Plk1 is capable of suppressing Plk1 expression, and therefore, significantly inhibits cellular proliferation, induces cell cycle arrest and apoptosis. Moreover, the sensitivity of lung cancer cells to chemotherapy is increased.     

Construction and expression of reconstructive plasmids with human thrombomodulin gene

AIM: To provide experimental evidence for gene therapy of thrombophilia disease, we constructed the eukaryotic expression plasmid with human thrombomodulin (hTM) gene and observed the alteration of hTM expression on the surface of human umbilical vein endothelial cells (HUVECs) with and without the reconstructive plasmid. METHODS: The whole expressive fragment of hTM gene was amplified by PCR from human genome. Both hTM gene and pcDNA3.1(+)/neo empty vector was digested by HindⅢ and EcoRⅠ. Two digested fragments were ligated into pcDNA3.1/hTM with T₄DNA ligase. After identifying, the reconstructive plasmid transfected into HUVECs using lipofectin. The hTM antigen on the HUVECs was detected by immunohistochemistry. RESULTS: The hTM reconstructive plasmid was confirmed by double endonuclease redigesting and sequencing. About 10% HUVECs were transfected by pcDNA3.1/hTM plasmid with lipofectin and the high-level hTM was detected on the transfected cells. CONCLUSION: We constructed the pcDNA3.1/hTM plasmid successfully, and it could be expressed on the HUVECs.     

NKX3.1 down-regulates bcl-2 gene expression in prostate cancer PC-3 cells

AIM: To investigate the effect of NKX3.1 on the gene expression of bcl-2 and apoptosis in prostate cancer PC-3 cells. METHODS: The eukaryotic expression plasmid pcDNA3.1- NKX3.1 was transiently transfected into PC-3 cells. RT-PCR and Western blotting were used to detect the effects of NKX3.1 on the expression of bcl-2 gene. Down-regulatory effect of NKX3.1 on bcl-2 gene was detected by EMSA. Flow cytometry and apoptotic body staining were carried out to study the effects of NKX3.1 on apoptosis of PC-3 cells. RESULTS: The mRNA and protein expression of bcl-2 in PC-3 cells was down-regulated by over-expression of NKX3.1. The EMSA result showed that NKX3.1 interacted with the NKX3.1 binding elements in upstream regulatory region of bcl-2 gene. The results of flow cytometry showed that the number of apoptotic PC-3 cells increased by 1.41-fold after NKX3.1 transfection to PC-3 cells. NKX3.1 increased the apoptotic bodies stained by Hoechst 33258 significantly. CONCLUSION: NKX3.1 down-regulates the expression of anti-apoptotic gene bcl-2 and induces the apoptosis of prostate cancer PC-3 cells.     

Effects of FKBP12.6 gene on intracellular Ca2+ concentration in mouse H9c2 (2-1) myocardial cells transfected by ultrasound-mediated destruction of microbubbles

AIM:To investigate the changes of intracellular calcium ion (Ca²⁺) concentration in mouse H9c2 (2-1) cells transfected with or without FK506 binding protein 12.6(FKBP12.6) gene by ultrasound mediated destruction of microbubbles. METHODS:The pcDNA3.1-FKBP12.6 plasmid, mingled with albumin-coated microbubbles agents, was transfected into H9c2 (2-1) cells by ultrasound-mediated destruction of microbubbles. The H9c2 (2-1) cell growth state was investigated by inverted microscope. The changes of intracellular Ca²⁺ concentration was determined by laser scanning confocal microscope. The FKBP12.6 protein expression was checked by immunohistochemistry. RESULTS:As compared with control cells, the H9c2 (2-1) cells, transfected with FKBP12.6 gene, grew better, had higher gross intracellular Ca²⁺ concentration. CONCLUSION:FKBP12.6 gene augments Ca²⁺ concentration in mouse H9c2 (2-1) cells, enhances the contractibility of the myocardial cell, which may be helpful to improve the myocardial dysfunction.     

Effect of P53 binding site on regulation of human NOD8 gene

AIM: To investigated the characterization and biological function of P53 binding element in the regulation of NOD8 gene. METHODS: Using the method of bioinformatics, we found a completely preserved P53 binding site in NOD8 core promoter both in humans and chimpanzees. NOD8 gene was amplified by PCR from human cDNA and correctly connected into the vector p NOD8 (760 bp)-EGFP-C2/ mp NOD8 (750 bp)-EGFP-C2. The constructed plasmids p NOD8 (760 bp)-EGFP- NOD8 and mp NOD8 (750 bp)-EGFP- NOD8 were transiently transfected into the cell line HEK293 by JetPei^TM and treated with pifithrin alpha (PFT-α,an inhibitor of P53) at different concentrations for 24 h. The mRNA and protein expression levels of NOD8 were detected by RT-PCR and Western blotting. In addition, chromatin immunoprecipitation (ChIP) assay was performed to determine the binding of P53 to the NOD8 promoter after recombinant plasmid p NOD8 (760 bp)-EGFP was transfected into HEK293 cells. RESULTS: The plasmids p NOD8 (760 bp) -EGFP- NOD8 and mp NOD8 (750 bp)-EGFP- NOD8 were successfully constructed and conformed by restriction digestion and sequence analysis. The results of ChIP confirmed that P53 bound to the promoter of NOD8 . The mRNA expression of NOD8 in the cells transfected with p NOD8 (760 bp)-EGFP- NOD8 was stronger than that in the cells transfected with control vector pEGFP-C2 (P<0.05). Furthermore, the mRNA expression of NOD8 was reduced in HEK293 cells transfectnt with the mutant plasmid mp NOD8 (750 bp)-EGFP- NOD8 compared with the cells transfected with p NOD8 (760 bp)-EGFP- NOD8 . Meanwhile, PFT-α inhibited the mRNA expression of NOD8 in the cells transfected with p NOD8 (760 bp)-EGFP- NOD8 in a concentration-dependent manner, and PFT-α at concentration of 90 μmol/L was the most effective in inhibiting NOD8 mRNA expression (P<0.01). As expected, the protein expression of NOD8 in pNOD8 (760 bp)-EGFP- NOD8 group significantly increased compared with that in pNOD8 -C2 group, the protein expression of NOD8 in mp NOD8 (750 bp)-EGFP- NOD8 group or pNOD8 (760 bp)-EGFP- NOD8 + PFT-α group was dramatically decreased compared with that in p NOD8 (760 bp) -EGFP- NOD8 group. CONCLUSION: The results suggest that the P53 binding site is critical for the regulation of NOD8 gene and there is positive feedback regulation between P53 binding site and NOD8 , which may maintain efficient balance between defense and self-inflicted injury in response to the invasion of pathogen.