Biochemical characterization of the recombinant schistosome tegumental protein SmALDH_312 produced in E. coli and baculovirus expression vector system

BACKGROUND The heterologous expression of parasitic proteins is challenging because the sequence composition often differs significantly from host preferences. However, the production of such proteins is important because they are potential drug targets and can be screened for interactions with new lead compounds. Here we compared two expression systems for the production of an active recombinant aldehyde dehydrogenase (SmALDH_312) from Schistosoma mansoni, which causes the neglected tropical disease schistosomiasis. RESULTS We produced SmALDH_312 successfully in the bacterium Escherichia coli and in the baculovirus expression vector system (BEVS). Both versions of the recombinant protein were found to be active in vitro, but the BEVS-derived enzyme showed 3.7-fold higher specific activity and was selected for further characterization. We investigated the influence of Mg2+, Ca2+ and Mn2+, and found out that the specific activity of the enzyme increased 1.5-fold in the presence of 0.5 mM Mg2+. Finally, we characterized the kinetic properties of the enzyme using a design-of-experiment approach, revealing optimal activity at pH 7.6 and 41C. CONCLUSIONS Although, E. coli has many advantages, such as rapid expression, high yields and low costs, this system was outperformed by BEVS for the production of a schistosome ALDH. BEVS therefore rovides an opportunity for the expression and subsequent evaluation of schistosome enzymes as drug targets

Expression and immunization assessment of HSV-2 glycoprotein D in baculovirus expression vector system / 吉林大学学报(医学版)

Objective:To express the glycoprotein D of herpes simplex virus type 2 (gD2) in the insect cells,and to determine its immunogenicity.Methods:HSV-2 genome was used as the template for amplification of gD2 extracellular domain fragment gene by PCR.The PCR product was inserted into the vector Bacmind,and the constructed recombinant plasmid gD2-Bacmind was transfected into the sf9 cells to package the recombinant baculovirus.The Sf9 cells were infected by recombinant baculovirus seed derived from the forth passage(P4),the titer of P4 recombinant baculovirus was detected by a plaque assay and the expression of recombinant protein gD2 was determined by Western blotting method.The supernatant of infected cells was collected and purified by Ni-NTA affinity chromatography to obtain the target protein gD2,the purified gD2 protein was used to immunize the BALB/c mice in 0, 2, 4 weeks (gD2 group),and PBS was used as negative control(PBS group);the titers of gD2 specific IgG in serum were detected by ELISA assay.Results: The PCR analysis and sequencing results proved that gD2-Bacmind was constructed correctly.The titer of recombinant baculovirus was 2.0×109 pfu·mL-1,the purified gD2 was about 37 000 with expectation,the percentage of gD2 in total protein was 90%.The average value of Log10 of titer of gD2 specific IgG in serum detected by ELISA assay in gD2 group at the sixth week was 4.34,and there was significant difference compared with PBS group(P<0.01).Conclusion: The gD2 expressed by insect-baculovirus expression vector system has the immunogenicity and can be selected as candidate protein for HSV-2 vaccine.

Expression, purification and immunogenicity analysis of recombinant MPB64 using baculovirus expression system / 中国免疫学杂志

Objective:To express mycobacterium tuberculosis protein MPB 64 in baculovirus insect cell expression system ,and identify its immunogenicity.Methods:The target gene MPB64 was connected to pFastBac vector ,then the pFastBac-MPB64 plasmid which was harvested would transformed to DH10Bac competent,and the target gene was transposition into Bacmid by Tn7 transposase fragment,therefore Bacmid-MPB64 Shuttle vector was obtained.The shuttle vector was packaged by liposomes and transfected Sf 9 cells to harvest P1-generation virus ,then high titers of P4 generation virus was harvested by repeat transfected Sf 9 cells three times.The target protein MPB64 was purified from the supernatant by Q Sepharose FF and Ni affine chromatography ,which were used to immunize BALB/c mice.Antibody changes in serum would be detected ,and the proliferation of immunized mice spleen cells would be detected by MTT,detected the IFN-γsecretion by MPB64 stimulated spleen cells by ELISA method.Results: MPB64 successfully expressed in insect cells.The purity of target protein was over 90% and yield up to 35 mg/L after purification.Purified protein can effectively stimulate BALB/c mice to produce antibodies , increase the content of IFN-γmedium in mice spleen cells ,and significantly promoting proliferation in spleen cells between 0.2-100 μg/ml.Conclusion: MPB64 which has immunogenicity was successfully expressed in baculovirus insect cell expression system ,that open a new avenue for tuberculosis vaccine production.

Assessment of patients discomfort and nursing care with the application of vector system in periodontal treatment / 中国实用护理杂志

Objective The study aimed to compare the vector system with the hand instrument in the pain severity during scaling and root planning.Methods 60 periodontal patients were randomly divided into two groups for supportive periodontal therapy (SPT):the experimental group (using vector system) and the control group (using gracey instruments),with 30 cases in each group.And the painfulness after SPT was evaluated by Visual Analogue Scale (VAS).Results 66％ patients felt mild pain during the supportive periodontal therapy with the application of vector system,while 36％ patients felt mild pain in the control group.24 patients in the experimental group accepted vector system for SPT and 23 patients in the experimental group felt less fear of the treatment.Conclusions Patients will feel less discomfort with the application of the vector system,therefore better compliance will be reached.And the cooperation of the doctors and nurses has great impact on the treatment effect.

Construction of Salmonella Pullorum C79-13ΔcrpΔasd mutant balanced-lethal host-vector system and its biological features / 中国免疫学杂志

In order to develop a safer vaccine strain exploit Salmonella Pullorum vaccine strain as a live vaccine vector.Methods:AΔcrpΔasd mutant of S.pullorum C79-13 strain was constructed and it was developed E.coli donor strain mutant was generated through the two-step method introduced by χ7213 ( pREΔasd) was conjugated with the recipient of C 79-13Δcrp.The C79-13ΔcrpΔasd the transduction of recombinant suicide plasmid .Results:PCR and sequencing results showed that ΔsipBSL1344 was suc-cessfully constructed.The further study indicated that foreign DAP must be supplied for the ΔcrpΔasd mutant to grow,in addition,the asd gene was transmitted stably .Compared with C79-13 strain,the O antigens was identical to C79-13 strain,but the growth velocity was reduced significantly ,significantly reduced virulence .Conclusion: The ΔcrpΔasd mutant can accept asd+plasmid to construct host-vector balance lethal system , and this system can be used to express foreign gene efficiently and to develop potential oral multivalent vaccines.

Development of a Gene Therapy Method for Cervical Cancer Using Attenuated Coxsackievirus B3 as a Vector System

We previously reported the development of an attenuated coxsackievirus B3, known as YYFF, which functioned as a viral vector system for foreign gene expression. In this study, we demonstrated the potential use of YYFF as a gene therapy vector. Recombinant YYFF was constructed to express the human papillomavirus 16 (HPV16) E7 gene, referred to as YYFF-HPV16-E7. Growth of YYFF-HPV16-E7 resembled the wild type, YYFF, and it expressed HPV16-E7 in cell culture. When YYFF-HPV16-E7 was directly injected into TC-1-transplanted C57/BL6 mice, there was no reduction in tumor size, because of the non-growth of YYFF in C57/BL6 mice. However, when YYFF-HPV16-E7-induced immune cells/serum that originated from BALB/c mice was passively delivered into BALB/c background TC-1-transplanted nude mice, it reduced the size of cervical tumors in the nude mice. This study indicates the potential use of YYFF-HPV16-E7 as a gene therapy agent for treating HPV-induced cervical cancer.

Mouse Dual Ig Domain Containing Cell Adhesion Molecule Protein Expression and Purification Using the Baculovirus Expression Vector System

A baculovirus expression vector system (BEVS) is used routinely to produce recombinant proteins in the milligram scale. Dual Ig domain containing cell adhesion molecule (DICAM) belongs to the type I class of transmembrane proteins. It consists of a signal peptide, two V-type Ig domains in the extracellular region, and a short cytoplasmic tail of 442 amino acids. To purify the recombinant DICAM protein from cells overexpressing the mouse full-length DICAM gene, recombinant baculovirus is infected and recovered in the Sf9 cells. As a result, mouse DICAM protein was efficiently expressed and extracted from the insect cells using the BEVS. This recombinant protein can be used in further studies for functional test of DICAM protein in the cells.

Post-translational ligation and function of dual-vector transferred split CFTR gene / 药学学报

The mutation of cystic fibrosis transmembrane conductance regulator (CFTR) gene leads to an autosomal recessive genetic disorder cystic fibrosis (CF). The gene therapy for CF using adeno-associated virus (AAV) vectors delivering CFTR gene is restricted by the contents limitation of AAV vectors. In this study the split CFTR genes severed at its regulatory domain were delivered by a dual-vector system with an intein- mediated protein trans-splicing as a technique to investigate the post-translational ligation of CFTR half proteins and its function as a chloride ion channel. A pair of eukaryotic expression vectors was constructed by breaking the human CFTR cDNA before Ser712 codon and fusing with Ssp DnaB intein coding sequences. After co-transfection into baby hamster kidney (BHK) cells followed by transient expression, patch clamps were carried out to record the chloride current of whole-cell and the activity of a single channel, and the ligation of two halves of CFTR was observed by Western blotting. The results showed that the intein-fused half genes co-tansfected cells displayed a high whole cell chloride current and activity of a single channel indicating the functional recovery of chloride channel, and an intact CFTR protein band was figured out by CFTR-specific antibodies indicating that intein can efficiently ligate the separately expressed half CFTR proteins. The data demonstrated that protein splicing strategy could be used as a strategy in delivering CFTR gene by two vectors,encouraging our ongoing research program on dual AAV vector system based gene transfer in gene therapy for cystic fibrosis.

A New Reporter Vector System Based on Flow-Cytometry to Detect Promoter Activity

In this study, we report the development of a new dual reporter vector system for the analysis of promoter activity. This system employs green fluorescence emitting protein, EGFP, as a reporter, and uses red fluorescence emitting protein, DsRed, as a transfection control in a single vector. The expression of those two proteins can be readily detected via flow cytometry in a single analysis, with no need for any further manipulation after transfection. As this system allows for the simultaneous detection of both the control and reporter proteins in the same cells, only transfected cells which express the control protein, DsRed, can be subjected to promoter activity analysis, via the gating out of all un-transfected cells. This results in a dramatic increase in the promoter activity detection sensitivity. This novel reporter vector system should prove to be a simple and efficient method for the analysis of promoter activity.

CONSTRUCTION,EXPRESSION AND DETECTION OF THE EUKARYOTIC EXPRESSION VECTOR OF SEROTONIN N-ACETYLTRANSFERASE GENE / 解剖学报

Objective To investigate the possibility that the construction and expression of a eukaryotic expression vector system of rat NN-NAT gene. Methods The full-length cDNA fragment of rat AA-NAT gene was amplified by RT-PCR method.After retrieving the PCR products,ligating it with pTARGET~(TM) vector,transformating ligation reaction to JM109 huge efficiency competent cells and identifying the recombinant plasmid,the recombinant eukaryotic expression vector pTARGET~(TM)-AANAT was transfected into rat L6 myoblasts with lipofectamine.Accordingly,engineered cells selected by antibiotic G418 were detected by the methods of RT-PCR and Westem blotting. Results It was revealed that,amplified AA-NAT cDNA confirmed by agarose gel electrophoresis could ligate with pTARGET~(TM) vector and subcloned into JM109 cells.L6 cells transfected with pTARGET~(TM)-AA-NAT survived well after G418 selection and expressed AA-NAT protein. Conclusion Our results suggest that we have prepared rat AA-NAT stable eukaryotic expression system successfully although it was just a primary result.This system can be used for the transfection of L6 myoblast.

Studies of killing and bystander effect of tumor necrosis factor related apoptosis-inducing ligand gene on hepatic carcinoma cell / 中华消化杂志

Objective To study the bystander effect of tumor necrosis factor related apoptosis inducing ligand(TRAIL) gene and it's mechanism. Methods Full length cDNA of human TRAIL was transfected into SMMC7721 with binary adenoviral vectors system. RT PCR was used to determine the expression of TRAIL gene. By MTT assay the effects of the TRAIL gene on the proliferation of SMMC7721 was studied; The apoptosis inducing ability of TRAIL gene on SMMC7721 was tested by fluorescence activated cell sorting (FACS). Bystander effect by testing the proliferation of the mixed cells of SMMC7721/TRAIL and SMMC7721 with different ratios, and the mechanism of bystander effect by testing the proliferation of SMMC7721 cultured with media of SMMC7721/TRAIL without cell components were also studied. Results TRAIL gene expressed by binary adenoviral vector system was able to inhibit proliferation(91.2%) and induce apoptosis(29.07%) of SMMC7721, significant difference between TRAIL gene and the other three controls were observed(PBS, LacZ, Bax)( P