Recombinant Eukaryotic Expression Plasmid Research Articles

Establishment of an Indirect ELISA Method for the Detection of the Bovine Rotavirus VP6 Protein.

The objective of this study was to develop an indirect ELISA utilizing a polyclonal antibody against bovine rotavirus (BRV) VP6 protein. To achieve this, pcDNA3.1-VP6, a recombinant eukaryotic expression plasmid, was constructed based on the sequence of the conserved BRV gene VP6 and was transfected into CHO-K1 cells using the transient transfection method. The VP6 protein was purified as the coating antigen using nickel ion affinity chromatography, and an indirect ELISA was subsequently established. The study found that the optimal concentration of coating for the VP6 protein was 1 μg/mL. The optimal blocking solution was 3% skim milk, and the blocking time was 120 min. The secondary antibody was diluted to 1:4000, and the incubation time for the secondary antibody was 30 min. A positive result was indicated when the serum OD450 was greater than or equal to 0.357. The coefficients of variation were less than 10% both within and between batches, indicating the good reproducibility of the method. The study found that the test result was positive when the serum dilution was 217, indicating the high sensitivity of the method. A total of 24 positive sera and 40 negative sera were tested using the well-established ELISA. The study also established an indirect ELISA assay with good specificity and sensitivity for the detection of antibodies to bovine rotavirus. Overall, the results suggest that the indirect ELISA method developed in this study is an effective test for detecting such antibodies.

Bovine viral diarrhea virus Erns protein expressed in Chinese hamster ovary cells and its immunogenicity analysis

The aim of this study was to produce Erns protein of bovine viral diarrhea virus (BVDV) by using suspensively cultured CHO cells expression system and to analyze the immunogenicity of the purified Erns protein. In this study, the recombinant eukaryotic expression plasmid pcDNA3.1-BVDV-Erns was constructed based on the gene sequence of BVDV-1 NADL strain. The Erns protein was secreted and expressed in cells supernatant after transfecting the recombinant expression plasmid pcDNA3.1-BVDV-Erns into CHO cells. The expression and purification of the Erns protein was analyzed by SDS-PAGE, the reactivity was determined with anti-His monoclonal antibodies and BVDV positive serum with Western blotting. Immunogenicity analysis of the Erns protein was determined after immunizing New Zealand white rabbits, and the serum antibodies were tested by indirect ELISA (iELISA) and indirect immunofluorescence (IFA). The serum neutralizing titer of the immunized rabbits was determined by virus neutralization test. The concentration of the purified Erns protein was up to 0.886 mg/mL by BCA protein quantification kit. The results showed that the Erns protein could be detected with anti-His monoclonal antibodies and anti-BVDV sera. Serum antibodies could be detected by iELISA on the 7th day post-prime immunization, and the antibody level was maintained at a high titer until the 28th day post-immunization. The antibody titer was 1:128 000. Furthermore, the expression of the Erns protein in BVDV-infected MDBK cells could be detected with immunized rabbits sera by IFA. Moreover, antigen-specific neutralizing antibodies of 2.71 log10 was induced in rabbits. In this study, purified BVDV Erns protein was successfully produced using CHO suspension culture system, and the recombinant protein was proved to have a good immunogenicity, which may facilitate the development of BVD diagnosis method and novel subunit vaccine.

Recombinant porcine interferon-gamma expressed in CHO cells and its antiviral activity

The aim of this study was to produce recombinant porcine interferon gamma (rPoIFN-γ) by Chinese hamster ovarian (CHO) cells expression system and to analyze its antiviral activity. Firstly, we constructed the recombinant eukaryotic expression plasmid pcDNA3.1-PoIFN-γ and transfected into suspension cultured CHO cells for secretory expression of rPoIFN-γ. The rPoIFN-γ was purified by affinity chromatography and identified with SDS-PAGE and Western blotting. Subsequently, the cytotoxicity of rPoIFN-γ was analyzed by CCK-8 test, and the antiviral activity of rPoIFN-γ was evaluated using standard procedures in VSV/PK-15 (virus/cell) test system. Finally the anti-Seneca virus A (SVA) of rPoIFN-γ activity and the induction of interferon-stimulated genes (ISGs) and cytokines were also analyzed. The results showed that rPoIFN-γ could successfully expressed in the supernatant of CHO cells. CCK-8 assays indicated that rPoIFN-γ did not show cytotoxicity on IBRS-2 cells. The biological activity of rPoIFN-γ was 5.59×107 U/mg in VSV/PK-15 system. Moreover, rPoIFN-γ could induced the expression of ISGs and cytokines, and significantly inhibited the replication of SVA. In conclusion, the high activity of rPoIFN-γ was successfully prepared by CHO cells expression system, which showed strong antiviral activity on SVA. This study may facilitate the investigation of rPoIFN-γ function and the development of novel genetically engineered antiviral drugs.

Eukaryotic expression and immunogenicity of Ancylostoma ceylanicum calreticulin.

Ancylostoma ceylanicum is a zoonotic soil-derived nematode that parasitizes human and animal intestines, causing malnutrition and iron-deficiency anemia. Calreticulin is a multifunctional protein involved in all stages of parasitic infection. Studies have found that parasites can secret calreticulin to regulate the host's immune response. To explore the immunogenicity of the eukaryotic expression plasmid of Ancylostoma ceylanicum calreticulin (Ace-CRT), we constructed a recombinant Ace-CRT eukaryotic expression plasmid (pEGFP-N3-Ace-CRT). Successful expression of the target protein in Human Embryonic Kidney (HEK) 293T cells was confirmed by indirect immunofluorescence and Western blot analysis. BALB/c mice were immunized with pEGFP-N3-Ace-CRT plasmid. Measuring IgG antibody levels in immunized mice sera by ELISA showed that the recombinant plasmid stimulated IgG antibody production in mice. Spleen lymphocytes were collected from vaccinated mice to determine the proportion of T cell subsets and the expression levels of cytokines. Flow cytometry revealed that the percentage of CD3+CD4+ and CD3+CD8+ T cells in mice spleen in the immunization group was significantly higher than that in the control group. Recombinant plasmid immunization increased IL-4, IL-10, IL-12, and IL-13 expression while decreasing IL-5, IL-6, and INF-γ in mice spleens. These results indicate that the eukaryotic plasmid constructed in this study had good immunogenicity and mainly induced a T helper 2 response in the host, laying a foundation for screening candidate molecules for anti-hookworm vaccines.

Transepithelial Secretion of Mucosal IgM Mediated by Polymeric Immunoglobulin Receptor of Flounder (Paralichthys olivaceus): In-Vivo and In-Vitro Evidence.

Secretory immunoglobulin (SIg) is crucial for mucosal surface defenses, but the transepithelial secretion of SIg mediated by polymeric immunoglobulin receptor (pIgR) is not clarified in fish. We previously found that flounder (Paralichthys olivaceus) pIgR (fpIgR) and secretory IgM (SIgM) increased in gut mucus post-vaccination. Here, the fpIgR-positive signal was mainly observed in the intestinal epithelium, whereas the IgM-positive signal was mainly distributed in the lamina propria, before immunization. IgM signals increased in the lamina propria and then in the epithelium after immunization with inactivated Vibrio anguillarum, and co-localization between IgM and fpIgR in the epithelium was determined, while the presence of EdU+IgM+ cells in the lamina propria identified the proliferative B cells, revealing that the secretion and transepithelial transport of SIgM locally occurred in the gut of flounder. Subsequently, we established an in-vitro model of transfected MDCK cells that stably expressed the fpIgR. After a recombinant eukaryotic expression plasmid (pCIneoEGFP-fpIgR) was constructed and transfected into MDCK cells, stable expression of the fpIgR in transfected MDCK-fpIgR cells was confirmed, and the tightness and integrity of the polarized cell monolayers grown on Transwells were evaluated. Afterward, the serum IgM of flounder was purified as a binding ligand and placed in the lower compartment of Transwells. An ~800-kDa protein band in the upper compartment was shown to be IgM- and fpIgR-positive, and IgM-positive fluorescence was seen in MDCK-fpIgR cells but not in MDCK-mock cells. Hence, the fpIgR helped polymeric IgM to pass across MDCK-fpIgR cells via transcytosis in a basolateral-to-apical fashion. These new findings provide a better understanding of the pathways shaping mucosal IgM responses and the local mucosal immune mechanisms in teleosts.

Experimental study on anti angiogenesis of recombinant mouse angiostatin gene in mice with gallbladder carcinoma.

Gallbladder cancer is one of the gastrointestinal tumors with an extremely poor prognosis. Its incidence rate is gradually increasing worldwide, and the rate of radical resection surgery is extremely low. Not sensitive to radiotherapy and chemotherapy, with a very poor prognosis. This study aimed to investigate whether the recombinant mouse angiostatin gene transfected anti-angiogenic gallbladder cancer cells can express angiostatin protein with the activity of inhibiting the growth of vascular endothelial cells and the inhibitory effect on the growth of gallbladder cancer. The recombinant mouse angiostatin gene eukaryotic expression plasmid was transfected into the gallbladder cancer cell line by applying liposome LIPOFECTAMINE 2000, and its activity was detected by vascular endothelial cell proliferation analysis. The results show that angiostatin can inhibit the growth of transplanted gallbladder cancer, and as the number of injections increases, the inhibition rate of gallbladder cancer growth also increases. At the end of the experiment, the total inhibition rate of gallbladder cancer growth reached 95% 5%, 20%, 30%, 40% gradually increase. Therefore, angiostatin has potential clinical application value in gene therapy of gallbladder cancer.

Designing of DNA Vaccine Based on a Secretory Form of Major Capsid Protein of Human Papillomavirus Type 18

More than 99% of cervical cancers are associated with human papillomaviruses (HPVs) worldwide. Current HPV vaccines are safe, highly immunogenic, with effective immunity against specific HPV types. However, DNA vaccines are a new appealing platform which can be considered for designing the HPV vaccines. This study aimed to construct a recombinant eukaryotic expression plasmid containing L1 of HPV-18, tissue plasminogen activators (tPA), and pan HLA DR-binding epitope (PADRE) genes into the pVAX1 vector. The L1, tPA, and PADRE genes were amplified in a thermocycler. The polymerase chain reaction (PCR) products were cloned and insertion of the genes was confirmed using colony PCR, restriction enzymes analysis, and sequencing methods. Indirect immunofluorescence, RT-PCR, and western blot assays were applied to identify the target gene in HEK-293 cells. Total IgG and its isotypes in immunized mice were measured by enzyme-linked immunosorbent assay technique. Western blot analysis showed a protein band of about 67.5 kDa in supernatant and cell lysate of transfected cells. The results of mice immunization with different constructs (group 1: the pVAX-L1, group 2: pVAX-tPA-PADRE-L1, group 3: pVAX1, and group 4: PBS as controls) indicated that the pVAX1-tPA-PADRE-L1 construct induced a significantly higher level of total IgG than pVAX1-L1 (p=0.003). In conclusion, pVAX1-tPA-PADRE-L1 recombinant plasmid is a highly immunogenicconstruct and suggests as a promising candidate for vaccine development against HPV type 18 in low-middle-income countries.

Molecular cloning and expression of hosphoenolpyruvate carboxykinase dominant epitope in Leishmania infantum

Objective Based on the (phosphoenolpyruvate carboxykinase, PEPCK) dominant epitopes gene of Leishmania infantum that has been analyzed and selected, we construct the corresponding recombinant prokaryotic expression vector to obtain the recombinant protein, construct the corresponding recombinant eukaryotic expression vector and verify the expression of eukaryotic vector in NIH3T3 cells, which laid the foundation for the subsequent vaccination and infection of animal experiment. Methods Based on the PEPCK dominant epitopes gene sequence, the recombinant prokaryotic and eukaryotic expression plasmid pET32a- PEPCK and pVAX1- EPEPCK were constructed by PCR reaction and enzyme reaction and were respectively transfected into E. coli and NIH3T3 cells of mice for expression. The expression of prokaryotic recombinant plasmid and the prokaryotic expressed protein purified by HisTrap affinity column were detected through SDS-PAGE and Western-Blot, and the expression of pVAX1-E PEPCK was verified by immunofluorescence. Results The correct sequencing results of the recombinant prokaryotic and eukaryotic vectors showed that the construction of recombinant vectors was successful. The positive results of 48.08 kD of recombinant protein were detected in SDS-PAGE and Western Blot, and immunofluorescence results of NIH3T3 cells transfected with eukaryotic recombinant vectors showed positive results. Conclusion We successfully constructed the recombinant prokaryotic and eukaryotic expression vectors: pET32a- PEPCK and pVAX1-E PEPCK , successfully got the recombinant proteins that followed by proteinic purification and confirmed the expression of pVAX1-E PEPCK in NIH3T3 cells, which is the basis for experiments where animals are vaccinated with a DNA prime-protein boost vaccines. 摘要：目的 基于前期分析并选取的婴儿利什曼原虫 PEPCK 的优势表位基因, 构建相应重组原核表达载体以获得 重组蛋白, 构建相应重组真核表达载体并验证真核载体在 NIH3T3 细胞中的表达, 为后续动物的免疫和感染实验备下 基础。 方法 根据 PEPCK 优势表位基因序列, 经 PCR 反应及酶切连接构建重组原核与真核表达质粒 pET32a- PEPCK 和 pVAX1-E PEPCK 并分别转染至 E. coli 和 NIH3T3 细胞中进行表达。采用 SDS-PAGE 电泳和 Western Blot 鉴定原核重 组质粒在大肠杆菌中的表达和原核表达蛋白被镍柱纯化后的情况, 采用免疫荧光实验验证真核重组质粒在细胞中的 表达。 结果 重组原核与真核载体的正确测序结果表明重组载体构建成功。SDS-PAGE 电泳和 Western Blot 的结果 显示, 大肠杆菌中表达的原核重组蛋白以及纯化后的蛋白在 48.08 kD 处存在条带, 转染了真核重组载体的 NIH3T3 细胞 的免疫荧光结果呈阳性。 结论 成功构建了 PEPCK 优势表位基因的重组原核和真核表达载体: pET32a- PEPCK 和 pVAX1-E PEPCK , 成功表达相应的重组蛋白并纯化并验证了 pVAX1-E PEPCK 在 NIH3T3 细胞中的表达, 为后续 DNA 疫苗初次免疫和蛋白疫苗加强免疫的动物实验备下基础。

Mechanism of Telomerase Reverse Transcriptase Regulating Dendritic Cell Immune Function in Proliferation and Cell Cycle of Renal Cell Carcinoma

To select human renal cell carcinoma cell lines VMRC-RCZ After resuscitation, culture and passage, the cell concentration was adjusted to 1 × 106/ml, and the experiment was divided into a control group, a renal cancer cell + DC group (DC group), and a renal cancer cell + DC+ hTERTtransfection group (transfection Group), a total of 3 groups; the recombinant eukaryotic expression plasmid hTERT-IRES2-EGFP was constructed and transfected into mature DC; the renal cancer cell + DC group was transfected with the empty plasmid, and the renal cancer cell + DC+ hTERT transfection group was transfected with the overexpression plasmid The transfection efficiency was detected by RT-PCR method. Cultured for 12 h and 24 h respectively, the percentage of HLA-DR, CD40 and MHC-II molecules in three groups of DC immunophenotypes was determined according to flow cytometry assay, and the cytokines IL-12 and TNF-α expression level, DC apoptosis rate was detected by in situ hybridization, the rate of tumor cell proliferation was measured according to MTT method, and cell cycle was determined through flow cytometry.

Construction and Activity Analyses of Single Functional Mouse Peroxiredoxin 6 (Prdx6).

IntroductionPeroxiredoxin 6 (Prdx6) is a bifunctional protein with glutathione peroxidase activity and phospholipase A2 activity. Previous studies have shown a significant positive correlation between the intracellular survival ability of Brucella and Prdx6. Here, the Prdx6 enzyme with a single activity was constructed to facilitate study of the relationship between the single function of Prdx6 and Brucella infection.Material and MethodsThe target open reading frame (ORF) DNAs of Prdx6 with a single active centre were prepared using gene splicing by overlap extension PCR (SOE-PCR), and the recombinant eukaryotic expression plasmids inserted by Prdx6 with the single activity centre were constructed and transfected into murine Raw264.7 macrophages. The glutathione peroxidase activity and phospholipase A2 activity of the constructed Prdx6 were examined.ResultsThe core centres (Ser32 and Cys47) of Prdx6 were successfully mutated by changing the 94th nucleotide from T to G and the 140th nucleotide from G to C in the two enzyme activity cores, respectively. The constructed recombinant plasmids of Prdx6 with the single active centre were transfected into murine macrophages showing the expected single functional enzyme activity, which MJ33 or mercaptosuccinate inhibitors were able to inhibit.ConclusionThe constructed mutants of Prdx6 with the single activity cores will be a benefit to further study of the biological function of Prdx6 with different enzyme activity.

Influence of human recombinant eukaryotic expression plasmid pIRES-bone morphogenetic proteins 2-vascular endothelial growth factor 165 in vitro transfection on osteogenic differentiation of bone marrow mesenchymal stem cells

Objective To observe the influence of human recombinant eukaryotic expression plasmid pIRES-bone morphogenetic proteins 2 (BMP2)-vascular endothelial growth factor 165 (VEGF165) in vitro transfection on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Methods The isolated rabbit BMSCs were used as the research objects, and the recombinant plasmid pIRES-BMP2-VEGF165 was transfected into cells in the presence of liposomes. Then, G418 was used to screen stable cell lines in vitro and mRNA and protein levels were detected for transfection efficiency. Stable cell lines were then cultured and the cells were divided into pIRES group, pIRES-BMP2-VEGF165 transfection group and osteogenic induction group. Western blotting was used to detect the expression of Collagen Ⅰ (ColⅠ) and osteocalcin (OCN) protein at 7th and 21st day after cultivation, respectively. After 14 days of cultivation, gomori modified calcium and cobalt staining was used to detect alkaline phosphatase activity. After 21 days of cultivation, calcified nodules of cells were detected by alizarin red method calcium staining. Results Compared with the empty plasmid pIRES group, the expression of BMP2 and VEGF165 was detected at the mRNA and protein levels of pIRES-BMP2-VEGF165 plasmid group. Compared with empty plasmid group and osteogenic induction group, the expression levels of ColⅠ and OCN protein in recombinant plasmid group were significantly increased (Col Ⅰ: 0.597±0.040 vs. 0.230±0.020, P=0.000; 0.597±0.040 vs. 0.457±0.035, P=0.005; OCN: 0.433±0.038 vs. 0.083±0.021, P=0.000; 0.433±0.038 vs. 0.350±0.036, P=0.046). Positive relative area (%) of alkaline phosphatase staining increased significantly (69.000±5.568 vs. 18.333±3.056, P=0.000; 69.000±5.568 vs. 43.333±5.508, P=0.002). Positive relative area (%) of calcified nodules increased markedly (37.747±3.763 vs. 1.587±0.972, P=0.000; 37.747±3.763 vs. 19.330±4.033, P=0.008). Conclusion Human recombinant eukaryotic expression plasmid pIRES-BMP2-VEGF165 was successfully transfected into BMSCs. As compared with osteogenic induction group, the recombinant plasmid pIRES-BMP2-VEGF165 was more effective in osteogenic differentiation of the cells. Key words: Bone mesenchymal stem cells; pIRES-bone morphogenetic proteins 2 -vascular endothelial growth factor 165 plasmid; Transfection in vitro; Bone morphogenetic proteins 2; Vascular endothelial growth factor 165

Formulation in DDA-MPLA-TDB Liposome Enhances the Immunogenicity and Protective Efficacy of a DNA Vaccine against Mycobacterium tuberculosis Infection.

Despite the vaccine Mycobacterium bovis Bacillus Calmette–Guérin is used worldwide, tuberculosis (TB) remains the first killer among infectious diseases. An effective vaccine is urgently required. DNA vaccine has shown prophylactic as well as therapeutic effects against TB, while its weak immunogenicity hinders the application. As a strong inducer of Th1-biased immune response, DMT, consisting of dimethyldioctadecylammonium (DDA) and two pattern recognition receptor agonists monophosphoryl lipid A and trehalose 6,6′-dibehenate (TDB), was a newly developed liposomal adjuvant. To elucidate the action mechanism of DMT and improve immunological effects induced by DNA vaccine, a new recombinant eukaryotic expression plasmid pCMFO that secretes the fusion of four multistage antigens (Rv2875, Rv3044, Rv2073c, and Rv0577) of Mycobacterium tuberculosis was constructed. pCMFO/DDA and pCMFO/DMT complexes were then prepared and their physicochemical properties were analyzed. The immunogenicity and protection against M. tuberculosis infection in vaccinated C57BL/6 mice were compared. Formulation of DNA and two agonists into the DDA liposome decreased zeta potential but increased the stability of storage, which resulted in a slower and longer-lasting release of DNA from the DNA–DMT complex than the DNA–DDA liposome. Besides Th1-biased responses, pCMFO/DMT vaccinated mice elicited more significantly CFMO-specific IL2+ TCM cell responses in the spleen and provided an enhanced and persistent protection against M. tuberculosis aerosol infection, compared to pCMFO/DDA and pCMFO groups. Therefore, the adjuvant DMT can release DNA and agonists slowly, which might attribute to the improved protection of DMT adjuvanted vaccines. pCMFO/DMT, a very promising TB vaccine, warrants for further preclinical and clinical trials.

Plasmid pLXSN-Mediated Adrenomedullin Gene Therapy for Cerebral Vasospasm Following Subarachnoid Hemorrhage in Rats.

BackgroundThe aim of this study was to investigate the protective effect of ADM gene mediated by plasmid pVAX1 on cerebral vasospasm (CVS) after subarachnoid hemorrhage (SAH).Material/MethodsThe recombinant plasmid pVAX-ADM was successfully established, and 40 SD rats were randomly divided into normal saline, pVAX1, pVAX1-ADM low-dose, pVAX1-ADM mid-dose, and pVAX1-ADM high-dose groups. The circumference and diameter of basilar artery, diameter of middle cerebral artery and internal carotid artery, and thickness of basilar artery wall were observed. The levels of circulating endothelial cells (CEC) and levels of regional cerebral blood flow (rCBF) of the parietal cortex were detected at different time-points. The expression levels of serum ADM, ET-1, and NOS of each group and the neurological functions were compared.ResultsThe circumference and diameter of basilar artery and the diameter of the middle cerebral artery and internal carotid artery in pVAX1-ADM groups were significantly longer than those in the saline group and pVAX1 group (P<0.05), but the thickness of the basilar artery wall in pVAX1-ADM groups was significantly lower (P<0.05), and the levels of growth or decrease were both dose-dependent (P<0.05). Compared with the saline group and pVAX1 group, the expression levels of serum ADM, NOS, and rCBF in pVAX1-ADM groups were significantly higher (P<0.05), but the levels of serum ET-1 and CEC were significantly lower (P<0.05). The scores of neurobehavioral functions of pVAX1-ADM groups were significantly lower (P<0.05), and the scores were also dose-dependent (P<0.05).ConclusionsThe recombinant eukaryotic expression plasmid pVAX1-ADM can significantly relieve cerebral vasospasm, increase the expression of serum ADM and NOS, and decrease the expression of serum ET-1 in a rat model of CVS; it is dose-dependent and can also improve nervous system function.

Effects of interleukin-34 expressed by human bone marrow derived mesenchymal stem cells on THP-1 cells

To construct recombinant eukaryotic expression plasmid vector of human IL-34 gene, and to study the effects of IL-34 expressed by human bone marrow-derived mesenchymal stem cells (hBM-MSCs) on THP-1 cells. Full-length IL-34 encoding sequence was amplified by PCR. And this fragment was cloned into the plasmid pIRES2-EGFP. Western blotting and ELISA were used to analyze the expression of IL-34 in hBM-MSCs. THP-1 cells were cultured with hBM-MSCs medium containing IL-34 protein. Real-time PCR detected the effects of IL-34 on the expression of IL-10 and TNFα in THP-1 cells. Restrictive enzyme analysis and sequencing demonstrated that IL-34 eukaryotic expression vector was successfully constructed. IL-34 protein expressed by hBM-MSCs could promote IL-10 and TNFα expression in THP-1 cells. Those results show that IL-34 expressed by hBM-MSCs has regulating effect on THP-1 cells.

Knocking out of human DNAH2 gene in U2OS cells by CRISPR/Cas9n double nick system

To study the biological function of DNAH2 (Homo sapiens dynein, axonemal, heavy chain 2) gene, we constructed human stable U2OS cell line of DNAH2 gene knockout through CRISPR/Cas9n double nick system. The A, B sgRNAs (Single guide RNA) and complementary strands were designed and synthesized. The double-stranded structures were formed during annealing, and connected with BbsⅠ cohesive ends-containing pX462 linear vector to construct the recombinant eukaryotic expression plasmids, including pX462-DNAH2-A and pX462-DNAH2-B. After the co-transfection of the two plasmids into U2OS cells, the addition of puromycin and limiting dilution method were used to obtain positive monoclonal cell line. Western blotting assay was then performed to detect the expression of DNAH2 protein, and PCR-sequencing technology was finally utilized to analyze the mutation feature. The results showed that A, B sgRNAs duplex was successfully inserted into pX462 vector, and DNAH2 protein was not expressed and DNAH2 gene suffered from the frame-shift mutation in U2OS-DNAH2-KO monoclonal cell line. These demonstrated that DNAH2 knockout U2OS stable cell line was successfully constructed through CRISPR/Cas9n double nick system, which providing a useful tool for the study of DNAH2 gene.

Pig BVDV-2 non-structural protein (Npro) links to cellular antiviral response in vitro.

In this study, we constructed for the first time a full-length cDNA clone of pig-original bovine viral diarrhea virus 2 (BVDV-2) strain SH-28, modified the cDNA clone (pASH28) for mutant pASHΔNpro and derived virus strain vASHΔNpro by deleting the genomic region encoding the Npro polypeptide, and examined significance of protein Npro for antiviral responses in vitro. Data showed that Npro-deletion mutant virus vASHΔNpro led to significant overexpression of oligo adenylate synthetase (OAS), myxovirus-resistant protein 1 (Mx1), and ubiquitin-like protein 15 (ISG15). Data also revealed that overexpression of Npro, but not NS2 and NS3 proteins, resulted in significant down-regulation of OAS, Mx1, and ISG15 production (p≤0.05) in bovine cells as well as porcine cells transfected with Npro recombinant eukaryotic expression plasmids. Npro (but not NS2 and NS3) was also found to inhibit poly(IC) from inducing production of type I interferon (IFN-I). These results indicated that protein Npro may play multiple roles in regulating antiviral response in host cells interfered by pig BVDV-2 strain, and provided useful information to understand better the mechanism of BVDV-2 persistent infection in pigs.

Analysis of nuclear localization and signal function of MITF protein predisposing to Warrdenburg syndrome

To study the role of dysfunction of nuclear localization signals (NLS) of MITF protein in the pathogenesis of Waardenburg syndrome. Eukaryotic expression plasmid pCMV-MITF-Flag was used as a template to generate mutant plasmid pCMV-MITF△NLS-Flag by molecular cloning technique in order to design the mutagenic primers. The UACC903 cells were transfected transiently with MITF and MITF△NLS plasmids, and the luciferase activity assays were performed to determine their impact on the transcriptional activities of target gene tyrosinase (TYR). The oligonucleotide 5'-GAACGAAGAAGAAGATTT-3' was subcloned into pEGFP-N1 to generate recombinant eukaryotic expression plasmid pEGFP-N1-MITF-NLS. The NIH3T3 cells were transfected separately with MITF, MITF△NLS, pEGFP-N1 and pEGFP-N1-NLS plasmids, and their subcellular distribution was observed by immunoflorescence assays. Expression plasmids for the mutant MITF△NLS with loss of core NLS sequence and pEGFP-N1-NLS coupled with MITF△NLS were successfully generated. Compared with the wild-type MITF, MITF△NLS was not able to transactivate the transcriptional activities of promoter TYR and did not affect the normal function of MITF. MITF△NLS was only localized in the cytoplasm and pEGFP-N1 was found in both the cytoplasm and nucleus, whereas pEGFP-N1-NLS was mainly located in the nucleus. This study has confirmed the localization function of NLS sequence 213ERRRRF218 within the MITF protein. Mutant MITF with loss of NLS has failed to transactivate the transcriptional activities of target gene TYR, which can result in melanocyte defects and cause WS.

Radiosensitivity in HeLa cervical cancer cells overexpressing glutathione S-transferase π 1.

The aims of the present study were to investigate the effect of overexpressed exogenous glutathione S-transferase π 1 (GSTP1) gene on the radiosensitivity of the HeLa human cervical cancer cell line and conduct a preliminarily investigation into the underlying mechanisms of the effect. The full-length sequence of human GSTP1 was obtained by performing a polymerase chain reaction (PCR) using primers based on the GenBank sequence of GSTP1. Subsequently, the gene was cloned into a recombinant eukaryotic expression plasmid, and the resulting construct was confirmed by restriction analysis and DNA sequencing. A HeLa cell line that was stably expressing high levels of GSTP1 was obtained through stable transfection of the constructed plasmids using lipofectamine and screening for G418 resistance, as demonstrated by reverse transcription-PCR. Using the transfected HeLa cells, a colony formation assay was conducted to detect the influence of GSTP1 overexpression on the cell radiosensitivity. Furthermore, flow cytometry was used to investigate the effect of GSTP1 overexpression on cell cycle progression, with the protein expression levels of the cell cycle regulating factor cyclin B1 detected using western blot analysis. Colony formation and G2/M phase arrest in the GSTP1-expressing cells were significantly increased compared with the control group (P<0.01). In addition, the expression of cyclin B1 was significantly reduced in the GSTP1-expressing cells. These results demonstrated that increased expression of GSTP1 inhibits radiosensitivity in HeLa cells. The mechanism underlying this effect may be associated with the ability of the GSTP1 protein to reduce cyclin B1 expression, resulting in significant G2/M phase arrest.

Identification of Putative ORF5 Protein of Porcine Circovirus Type 2 and Functional Analysis of GFP-Fused ORF5 Protein.

Porcine circovirus type 2 (PCV2) is the essential infectious agent responsible for causing porcine circovirus-associated diseases in pigs. To date, eleven RNAs and five viral proteins of PCV2 have been detected. Here, we identified a novel viral gene within the PCV2 genome, termed ORF5, that exists at both the transcriptional and translational level during productive infection of PCV2 in porcine alveolar macrophages 3D4/2 (PAMs). Northern blot analysis was used to demonstrate that the ORF5 gene measures 180 bp in length and overlaps completely with ORF1 when read in the same direction. Site-directed mutagenesis was used to show that the ORF5 protein is not essential for PCV2 replication. To investigate the biological functions of the novel protein, we constructed a recombinant eukaryotic expression plasmid capable of expressing PCV2 ORF5. The results show that the GFP-tagged PCV2 ORF5 protein localizes to the endoplasmic reticulum (ER), is degraded via the proteasome, inhibits PAM growth and prolongs the S-phase of the cell cycle. Further studies show that the GFP-tagged PCV2 ORF5 protein induces ER stress and activates NF-κB, which was further confirmed by a significant upregulation in IL-6, IL-8 and COX-2 expression. In addition, five cellular proteins (GPNMB, CYP1A1, YWHAB, ZNF511 and SRSF3) were found to interact with ORF5 via yeast two-hybrid assay. These findings provide novel information on the identification and functional analysis of the PCV2 ORF5 protein and are likely to be of benefit in elucidating the molecular mechanisms of PCV2 pathogenicity. However, additional experiments are needed to validate the expression and function of the ORF5 protein during PCV2 infection in vitro before any definitive conclusion can be drawn.

Upregulation of GRIM-19 inhibits the growth and invasion of human breast cancer cells.

Gene associated with retinoid-interferon (IFN)-induced mortality 19 (GRIM-19), a novel IFN-β/retinoic acid-inducible gene product, has been identified as a potential tumor suppressor, which is associated with the inhibition of tumor growth. GRIM-19 has been demonstrated to be downregulated in the ovarian tissue of patients with breast cancer, however, its role in breast cancer remains to be fully elucidated. In the present study, a recombinant eukaryotic expression plasmid carrying GRIM-19 was constructed and then transfected into the MCF7 human breast cancer cell line to examine its effects on breast cancer cell growth, migration and invasion using several in vitro approaches. The results demonstrated that upregulation GRIM-19 in the MCF7 cells significantly inhibited cell proliferation, colony formation, migration and invasion, and induced cell apoptosis. Additionally, upregulation of GRIM-19 also suppressed the secretion of urokinase-type plasminogen activator (u-PA), matrix metalloproteinase (MMP)-2, MMP-9 and vascular endothelial growth factor (VEGF). It was also demonstrated that the activation of signal transducer and activator of transcription 3 (STAT3) was downregulated by the expression of GRIM-19. These results revealed that overexpression of the GRIM-19 gene may be an effective approach to control the growth and invasion of human breast cancer cells.