Construction of PR domain eukaryotic expression vector and its inhibitory effect on esophageal cancer cells

Objective：PR domain is responsible for the tumor suppressing activity of RIZ1.The study aimed to construct human PR domain eukaryotic expression vectors,transfect human esophageal cancer cells （TE13）,and evaluate the anticancer activity of PR domain on human esophageal cancer TE13 cells.Methods：First,mRNA was extracted from human esophageal cancer tissue by RT-PCR,then reversetranscribed to cDNA.After amplifying from the DNA template,PR domain was linked to T vector.Second,after extraction,PR domain was cut using enzyme and linked to pcDNA3.1（＋）.Then,the plasmid was transfered to Trans1-T1 phage resistant competent cells,following by extracting the ultrapure plasmid,and transfecting into TE13 cells.In the end,the protein expression of pcDNA3.1（＋）/PR domain in TE13 was detected by Western blot,and the apoptosis of TE 13 by technique of flow cytometry.Results：More than 5,000 bp purposed band of pcDNA3.1（＋）/PR domain plasmid was found by agarose gel electrophoresis.After transfection,the PR domain （molecular weight of about 28 Da） was found only in 3,4 and 5 groups by Western blot.Flow cytometry assay showed apoptosis in experimental group was significantly more than that in the control group （P＜0.05）.Conclusions：The PR domain eukaryotic expression vector was constructed successfully.The protein of the PR domain could be expressed in esophageal cancer TE13 cells firmly after transfection,and a single PR domain could promote apoptosis of TE13 cells.

DNA immune responses induced by codelivery of IL-12 expression vectors with hepatitis C structural antigens

Objective: To demonstrate the utility of DNA vaccines for the tailored methods, the efficacy of enhanced immune responses, and the types of increased im- mune responses. Methods: Four recombinant plasmids constructed in- cluded the coding regions for the core protein (pC) and for the core, E_1 and E_2 together (pCE_1E_2), IL- 12 p35 and p40. These plasmids were transfected into mammalian cells to test their protein expression and were injected into the quadriceps muscles of BALB/ C mice for measurement of specific antibodies and cytotoxic T-lymphocyte (CTL) responses. Results: All the recombinant plasmids were shown to express specific antigens stably in mammalian cells. Codelivery of pIL-12 expression cassettes with pC and pCE_1E_2 in mice resulted in the enhancement of Ag-dependent CTL responses and the reduction of specific Ab response. The CTL activity was: pC= 18.65%±5.71%, pCE_1E_2=20.07%±11.11%, pC +pIL-12=60.11%±17.37%, pCE_1E_2+pIL-12= 67.48%±15.57%, respectively. The average A val- ues of anti-HCV were pC=0.415±0.127, pCE_1E_2= 0.358±0.096, pC+pIL-12=0.210±0.086, pCE_1E_2 +pIL-12=0.258±0.125. Conclusion: Codelivery of pIL-12 with plasmid DNA can enhance the efficacy of immune responses and shift the type of immune responses.

The inhibitory effects of siRNA expression vector on CXCR4 expression in prostate carcinoma cell lines

Objective： To investigate the inhibitory effects of RNAi （ RNA interference, RNAi） expression vector on CXCR4 expression in prostate carcinoma cell lines. Methods： Small interference RNA （siRNA） expression vectors for CXCR4 gene were constructed and transfected into prostate carcinoma cell lines（PC-3m and LNCaP）with liposomes. T expression of CXCR4 was detected by RT-PCR and western blot. Results： T expression of CXCR4 mRNA and protein in the PC-3m and LNCaP cells was reduced by RNAi expression vectors. The inhibitory rate of CXCR4 mRNA expression in the PC-3m cells was 87.81% ± 10.20% ,56.10% ± 9.32% at the 24th hour and the 48th hour, compared with 56.93% ±8.78% ,49.24% ± 11.23% in LNCaP cells. The inhibitory rate of the expression of CXCR4 protein was 64.71% ± 6.68% ,58.66% ± 11.56% respectively. Conclusion： The expression of CXCR4 gene can effectively be inhibited by RNAi expression vectors.

Construction and characterization of calreticulin-HBsAg fusion gene recombinant adenovirus expression vector

AIM: To generate recombinant adenoviral vector con-taining calreticulin (CRT)-hepatitis B surface antigen (HBsAg) fusion gene for developing a safe, effective and HBsAg-specific therapeutic vaccine.METHODS: CRT and HBsAg gene were fused using polymerase chain reaction (PCR), endonuclease diges-tion and ligation methods. The fusion gene was cloned into pENTR/D-TOPO transfer vector after the base pairs of DNA (CACC) sequence was added to the 5′ end. Adenoviral expression vector containing CRT-HBsAg fusion gene was constructed by homologous recombinan-tion. The human embryo kidney (HEK) 293A cells were transfected with linearized DNA plasmid of the recombi-nant adenoviral vector to package and amplify recombi-nant adenovirus. The recombinant adenovirus titer was characterized using the end-dilution assay. The expres-sion of the CRT/HBsAg fusion protein in Ad-CRT/HBsAg infected 293A cells was detected by Western blotting.RESULTS: The CRT-HBsAg fusion gene was char-acterized by PCR and sequencing and its length and sequence were confirmed to be accurate. The CRT-HB-sAg fusion gene recombinant pENTR/D-TOPO transfer vector was constructed. The recombinant adenoviral vector, Ad-CRT/HBsAg, was generated successfully. The titer of Ad-CRT/HBsAg was characterized as 3.9 × 1011 pfu/mL. The CRT-HBsAg fusion protein was ex-pressed by HEK 293A cells correctly. CONCLUSION: CRT/HBsAg fusion gene recombinant replication-defective adenovirus expression vector is constructed successfully and this study has provided an experimental basis for further studies of Hepatitis B vi-rus gene therapy.

Gene Therapy of HSV-TK Transferred by the EBV based Expression Vector on Experimental Hepatocellular Carcinoma

To study the therapeutic effects of herpes simplex virus thymidine kinase gene transferred by the EBV based expression vector on experimental hepatocellular carcinoma, pDR2 TK gene was delivered into human hepatocellular carcinoma cell line SMMC 7721 by using liposome mediated transfection technique,and then gene expression was detected by RT PCR, and the killing effects were examined through MTT method. In the nude mice hepatoma model,the antitumor effects of pDR2 TK /GCV system was evaluated in terms of tumor growth. MTT results showed that the pDR2 TK /GCV had cytotoxic effect and about 70 % SMMC 7721 cells were killed when GCV was at 1000 μmol/L. In vivo experiment showed that the tumor size in nude mice with transferred pDR2 TK gene was significantly smaller than that in control group . On the 10th day the tumor in 3 mice (60 %) disappeared completely after GCV treatment. It is concluded that the pDR2 TK/GCV system has marked killing effects on the experimental hepatocellular carcinoma.

Cloning of XET gene from Anthocephalus chinensis and its plant expression vector construction

A full-length cDNA sequence of xyloglucan endotransglycosylase gene （XET）, abundantly expressed in the cambium of Anthocephalus chinensis was cloned by conserved PCR, rapid-amplification of cDNA ends and by chromosome walking. Analytical results of the DNA sequence show that a 912 bp complete open reading frame （ORF） encoded a 303-amino acid protein was in the 1205 bp full cDNA sequence. The deduced amino acid sequence of AcXET, which contained the conserved specific EIDFE catalytic site sequence to XETs was homologous to the other known XET proteins. In order to study the gene function of AcXET and obtain transgenic plants, a plant expression vector pBIAcXET was constructed by recombinating the AcXET fragment from the cloning vector pMD19AcXET and the binary vector pBI121 between the XbaI and SmaI sites. The fragment ofAcXET gene was inserted between the CaMV 35S promotor and the coding region of the GUS gene in pBI121. The identification results show that the plant expression binary vector pBIAcXET was constructed successfully. These results lay the foundation for studying the molecular mechanism ofAcXET gene during wood formation.

Construction of Prokaryotic Expression Vector for pbv220/NT4-ADNF-9

Objective To construct a prokaryotic expression vector bearing fusion gene NT4-ADNF-9 for future studies on genetic therapies for sensorineural deafness. Methods Double strand ADNF-9 cDNA was synthesized using asymmetrical primer/ templates and ligated to the 3’ terminal of signal and leader peptides of neurotrophin 4 (NT4). The fusion gene NT4 -ADNF-9, was subcloned into prokaryotic expression vector pBV220, and named pBV220/ NT4-ADNF-9. DNA sequence of the fusion gene was analyzed. The fusion protein was isolated by SDS-PAGE and its bioactivity was evaluated using primary culture of day 8 chicken embryonic DRGcells. Results The correct sequence of fusion gene NT4-ADNF-9 was successfully subcloned into the pBV220 vector. The expressed ADNF-9 protein showed its effects in promoting cell survival and neurite growth. Conclusion Prokaryotic expression vector pBV220/NT4-ADNF-9 was constructed successfully and the expressed fusion protein demonstrated satisfactory bioactivity.

Construction of a Food-Grade Expression Vector Based on pMG36e by Using an α-Galactosidase Gene as a Selectable Marker

Construction of a food-grade expression vector for application to lactic acid bacteria（LAB） is of importance for dairy fermentation system. An α-galactosidase（aga） gene encoding an enzyme degrading melibiose was amplified by PCR from the plasmid p RAF800 of Lactococcus lactis NZ9000. The aga gene was introduced into pMG36 e to substitute the p rimary antibiotic selectable marker of pMG36 e, resulting in construction of a new food-grade expression vector pMG36-aga. To testify the expression efficiency of exogenous gene in pMG36-aga, a 1.5 kb long α-amylase（amy） gene from Ba cillus li cheniformis was cloned by PCR and introduced into the plasmid pMG36-aga. The resultant plasimd pMG36-aga-amy was transformed into L. lactis ML23 by electroporation. The positive clones were selected with the medium containing melibiose as the sole carbon source. Th e selection efficiency of aga was 8.71×103 CFU with a standard deviation of 9.1×102 CFU ？g-1 DNA of pMG36-aga. Furthermore, the SDS-PAGE analysis showed that the pMG36-aga-amy expressed a 56.4 kDa protein which was the same as the putati ve molecular weight of α-amylase. The starch plate assay also indicated that L. lactis ML23 displayed high activity of α-amylase by expressing of amy gene of pMG36-aga-amy.

Gene Cloning of Murine α-Fetoprotein Gene and Construction of Its Eukaryotic Expression Vector and Expression in CHO Cells

To clone the murine α fetoprotein (AFP) gene, construct the eukaryotic expression vector of AFP and express in CHO cells, total RNA were extracted from Hepa 1 6 cells, and then the murine α fetoprotein gene was amplified by RT PCR and cloned into the eukaryotic expression vector pcDNA3.1. The recombinant of vector was identified by restriction enzyme analysis and sequencing. After transient transfection of CHO cells with the vector, Western blotting was used to detect the expression of AFP. It is concluded that the 1.8kb murine α fetoprotein gene was successfully cloned and its eukaryotic expression vector was successfully constructed.

Effect of MSTN Propeptide and shRNA Co-expression Vector on Proliferation of Skeletal Muscle Satellite Cells

Myostatin （MSTN） is a negative regulator of skeletal muscle growth, in order to study the effect of inhibition MSTN expression on the proliferation of bovine skeletal muscle satellite cells, we constructed co-expression vector pcDNA3.1-Pro- MSTNshRNA, transfected it into muscle satellite cells by Liposome 2000, and detected cell proliferation changes by CCK-8 method and flow cytometry after 48 h. The expressions of P21 and CDK2 were detected by Western blot and real-time PCR. The results showed that the cell vitality of experimental groups significantly increased than that of the negative control, and cells in S phase also increased significantly （P〈0.05）. After knocked down MSTN gene, P21 expression decreased （P〈0.05）, but CDK2 gene expression increased （P〈0.05）. These results indicated that MSTN gene expression was associated with P21 and CDK2, the proliferation of skeletal muscle satellite cells could be promoted while MSTN was inhibited, which provided a theoretical basis for the study on transgenic cattle.

Construction of expression vector and transformation of FpDREB2A gene into Robinia pseudoacacia 'Idaho' mediated with Agrobacterium tumefaciens

The transcription factor gene FpDREB2A of Fraxinus pennsylvanica Marsh var. subintegerrima （Vahl.） Fern was con- structed into the higher plant expression vector pBin438 and transformed into Robinia pseudoacacia ‘Idaho＇ by Agrobacterium tu- mefaciens GV3101. Callus was screened with G418. Morphogenesis of shoots and roots of Idaho locust transformed genes was car- ried out on antibiotic media. The transformed plants were verified by PCR and Southern blotting tests that the FpDREB2A gene had been inserted into the genome DNA of Idaho locust.

Construction of expression vector for NT4-ADNF-9 fusion gene

Objective To construct the prokaryotic expression vector bearing fusion gene NT4-ADNF-9 and lay foundation for further study on genetic therapy of neurosensory deafness. Methods By means of asymmetrical primer/ template,double stranded cDNA of activity dependent neurotrophic factor-9 (ADNF-9) was obtained,which included restriction enzymes sites on the two extremities. ADNF-9 cDNA was ligated to the signal and leader peptides of neurotrophin 4 (NT4),and the fusion gene was named NT4-ADNF-9. Then it was subcloned into prokaryotic expression vector pBV220,and called pBV220/ NT4-ADNF-9. Results Evidences of DNA sequence analysis and restriction enzymes digestion showed that we recombined ADNF-9 cDNA to the 3’terminal of the signal and leader peptides of NT4,and the fusion gene was subcloned into pBV220 successfully. Bioactivity of the products was proved that it could support the cell survival and neurite growth in the primary cultures of dorsal root ganglia (DRG) of embryonic day-8 chicken neurons as compared to the control. Conclusion Prokaryotic expression vector pBV220/NT4-ADNF-9 can be constructed successfully and the bioactivity is satisfactory.

Construction and Identification of Mammary Gland-specific Expression Vector of Bovine Tracheal Antimicrobial Peptide (TAP)

[ Objective] This study aimed to construct nmnmm_ry gland-specific expression vector of bovine tracheal antimicrobial peptide （TAP） gene. [ Method] TAP gene of dairy cattle was amplified from the mammary gland tissue by RT-PCR using a pair of primers which were designed according to bovine TAP cDNA se- quence （NM_174776） in GenBank, and then cloned into pMD19-T Simple vector for sequencing. The recombinant plasmid was digested using EcoRI and KpnI, the target gene fragment was recovered and inserted into general mammary gland-specific expression vector pBLG-EGFP harboring enhanced green fluorescent protein （ EGFP）, and transfected into bovine mammary epithelial cells （bMEC）, COS-7 cells and lactating rabbit mmmnary gland tissue by lipofectin transfection. The ex- pression of green fluorescent protein in transfected cells was detected under fluorescence microscopy, and the expression of TAP mRNA in rabbit mammary gland tis- sue was detected by semi-quantity RT-PCR. [ Result] The constructed mammary gland-specific expression vector pBLG-EGFP-TAP specifically expressed EGFP in transfected bMECs. In addition, semi-quantitative RT-PCR result showed that the expression level of TAP mRNA in rabbit mammary gland tissue was significantly enhanced after transfeeted with pBLG-EGFP-TAP. [ Conclusion] The mammary gland-specific expression vector pBLG-EGFP-TAP was successfully constructed, which provided important materials for further investigation of expression characteristics of TAP gene and prevention of bovine mastitis by using genetic engineering technology.

Construction and Identification of Intestine-specific Expression Vector of Xylanase Gene(XynB)

The Aspergillus niger XynB gene and core promoter region of porcine RELMβgene were cloned into pcDNA3.1(-),and an intestine-specific expression vector pcDNA3.1-RELMβ-XynB-Myc-GFP carrying green fluorescence and Myc double tags was constructed.The vector was transfected into human colon cancer cells(HT29)and human liver cancer cells(Bel7402)using liposomes.Fluorescence microscopy revealed that the vector could specifically express green fluorescent protein(GFP)in HT29 cells.RT-PCR and Western Blot were performed on the HT29 cells transfected with the expression vector,and the results showed that the XynB gene was normally transcribed in HT29 cells,and the target protein expression was detected in the cells.

Construction of Plant Expression Vector of Heat Shock Factor Gene AtHsfA1a from Arabidopsis

[ Objective ] Heat shock factors （HSFs） are the major transcription factors of eukaryotic heat shock responses. This study aims to investigate the adversity stress tolerance functions of Arabidopsis heat shock factor AtHsfAla, which has important significance for in-depth understanding of adversity stress tolerance mechanisms of plants and further utilization of heat shock factor genes. [Method] Genomic DNA of Arabidopsis was extracted with CTAB method and purified to obtain Arabidopsis DNA samples for in vitro site-specific recombination cloning （ Gateway cloning） to construct plant expression vector of heat shock factor AtHs- fAla. Firstly, donor vector pDONR 201/AtHsfAla was constructed based on attB and attP site-specific recombination method （BP reaction）, to identify E. coli transformants harboring correct sequence of AtHsfAla by sequencing; secondly, plant expression vector pBTWG2/AttlsfAla overexpressing Arabidopsis heat shock factor AtHsfAla was constructed based on attL and attR site-specific recombination method （LR reaction）, to screen E. coli transformants harboring target plasmid. [ Result] Plant expression vector of Arabidopsis heat shock factor gene AtHsfAla was constructed successfully. [ Conclusion] This study not only provided experimental materials for acquiring transgenic plants overexpressing heat shock transcription factor AtHsfAla, but also laid the foundation for further investigation of the diversity of adversity stress tolerance functions reanlated by HSFs.

Cloning of CRABS CLAW Gene from Brassica napus and Construction of Its RNA Interference Expression Vector

CRABS ClAW （CRC） is a member of the YABBYA transcription factor gene family that plays an important role in floral organ development of plants. This study aimed to further investigate the regulatory function of CRC transcription factor in the development of floral organs of rape （Brassica napus L. ）. A 580 bp fragment of CRC gene was cloned by RT-PCR from total RNA of buds of rape cultivar Ningyou No. 10 to construct an inverted repeated expression cassette of CRC gene using intermediate vector pHturieane. Firstly, CRC gene fragment was positively inserted into the 5＇ end of a spliceable intron and negatively inserted into the 3＇ end of the intron. Subsequently, CaMV35S promoter sequence and inverted repeated expression cassette of CRC gene were transferred into pUC18 multiple clone site of binary expression vector pCAMBIAI1390. The constructed interference expression vector was named pA6-CRCi, which was further confirmed by restriction enzyme digestion and sequencing.