Gene therapy in neuromuscular disorders / Terapia gênica nas doenças neuromusculares

Abstract Monogenic neuromuscular disorders are potentially treatable through gene therapy. Using viral vectors, a therapeutic transgene aims to restore normal levels of a protein not produced by the defective gene, or to silence a gene whose expression leads to toxic effects. Spinal Muscular Atrophy (SMA) is a good example of a monogenic disease that currently has an AAV9-based vector gene therapy as a therapeutic option. In this review, we intend to discuss the viral vectors and their mechanisms of action, in addition to reviewing the clinical trials that supported the approval of gene therapy (AVXS-101) for SMA as well as neuromuscular diseases that are potentially treatable with gene replacement therapy.

Resumo Doenças neuromusculares monogênicas são potencialmente tratáveis através de terapia gênica. Utilizando-se de vetores virais, um transgene terapêutico objetiva repor os níveis normais de uma proteina não produzida pelo gene defeituoso ou silenciar um gene cuja expressão leva a efeitos tóxicos. A Atrofia Muscular Espinhal (AME) é um bom exemplo de doença monogenica que atualmente tem uma terapia gênica com vetor viral AAV9 como opção terapêutica. Nesta revisão, pretendemos discutir os vetores virais e macanismos de ação utilizados, além de revisar os ensaios clínicos que embasaram a aprovação da terapia gênica (AVXS-101) para AME, assim como doenças neuromusculares potencialmente tratáveis com terapia de reposição gênica.

Construction of the overexpressed lentiviral vector for targeting human TNFAIP1 gene / 中国医师杂志

Objective To construct a lentivirus vector over-expressing Tumor necrosis factor alpha induced protein 1 (TNFAIP1) gene and detect its expression level in vitro.Methods The full length of TNFAIP1 gene fragments were amplified by polymerase chain reaction (PCR).The pEZ-Lv105 vectors were digested by restriction endonuclease which was then linked to the full length of TNFAIP1 gene fragments by using T4DNA ligase.The plasmids were transfected into E.coli stbl3 and then we obtained the highly expressing positive clones by screening and identifying.The lentivirus vectors containing TNFAIP1 gene were transfected into 293T cells for package according to the packing kit manual.Results TNFAIP1 gene was amplified and successfully bound to the pEZ-Lv105 lentivirus vectors.The sequences of the recombinant plasmids were confirmed correctly by PCR and DNA sequence.The enhanced green fluorescent protein (eGFP) could be observed after recombinant lentiviruses were cotransfected into 293T cells.Conclusions The TNFAIP1 overexpressed lentivirus vector is successfully constructed,which provides a molecular tool for further study of TNFAIP1 gene in optic nerve glioma.

Experimental study of lentiviral vector-mediated bFGF gene transfection on rabbit BMSCs / 天津医药

Objective To observe the effect of lentiviral vector-mediated basic fibroblast growth factor (bFGF) gene transfection on the biological characteristics of rabbit bone marrow stromal cells(BMSCs)under in vitro culture conditions. Methods BMSCs were obtained by density gradient centrifugation and adherence screening. The bFGF gene was transfected into BMSCs by lentiviral vector and divided into bFGF transfection group,empty virus group and untransfected group according to the transfection conditions.After transfection,the morphology,expressions of bFGF mRNA and protein, cell proliferation,cell cycle and alkaline phosphatase(ALP)activity were observed in three groups of cells. Results High density BMSCs were successfully obtained by density gradient centrifugation and adherence screening.After transfection of BMSCs with bFGF gene, the cell morphology showed no significant changes, while the expressions of bFGF mRNA and protein were significantly increased, the cell proliferation curve shifted upward, the proportion of proliferating cells increased,and the activity of ALP was significantly enhanced.There were significant differences between three groups(P<0.05).Conclusion The rabbit bFGF gene is successfully introduced into the BMSCs cultured in vitro by lentiviral vector, and the target gene is stably expressed.The expression of bFGF can promote the proliferation and osteogenic differentiation of BMSCs.

Construction and identification of mouse Krüppel-like factor 4 recombinant lentiviral expression vector and its effect on proliferation of RAW264.7 cells / 天津医药

Objective To construct and identify a lentiviral vector carrying mouse Krüppel-like factor 4 (KLF4) gene, and establish RAW264.7 cell line of peritoneal macrophages that over-expressed KLF4. Methods KLF4 gene was cloned using the measure of polymerase chain reaction (PCR). Then the recombinant transfer vector pLVX-KLF4 (pLVX-KLF4-mCMV-ZsGreen-PGK-Puro) was constructed. The pLVX-KLF4 was confirmed through PCR, restriction enzyme digestion and sequencing. The correct recombinant transfer vector together with its two helper virus vectors (psPAX2 and pMD2.G) were cotransfected into the 293T cells by Lipofectamine? 3000. The supernatant of 293T was harvested to infect RAW264.7 cells. Flow cytometry (FCM) was used to test the viral titer of the expression level of green fluorescent protein. The expression of KLF4 mRNA in RAW264.7 cells was measured by real-time PCR. Results The restriction enzyme digestion, PCR and sequencing confirmed that the transfer lentiviral vector pLVX-KLF4 was constructed successfully. KLF4 mRNA was over-expressed in Lenti-KLF4 transfected RAW264.7 cells than that of wild type RAW264.7 cells (P<0.05). In transfected RAW264.7 cells, KLF4 mRNA was over-expressed (P<0.05). The recombinant lentivirus of KLF4(Lenti-KLF4)titer was 2.05×108 TU/mL measured by FCM.The flow cytometry results showed that the S phase fraction was prolonged and G0/G1 was arrested in the over-expressed KLF4 of RAW264.7 cells. The EdU showed that the up-regulated expression of KLF4 gene stimulated the proliferation of RAW264.7 cells. Conclusion The recombinant lentiviral vector, which can effectively express KLF4 mRNA, has been successfully constructed. The up-regulated KLF4 gene may increase the proliferation of RAW264.7 cells.

Gene therapy: advances, challenges and perspectives / Terapia gênica: avanços, desafios e perspectivas

ABSTRACT The ability to make site-specific modifications to the human genome has been an objective in medicine since the recognition of the gene as the basic unit of heredity. Thus, gene therapy is understood as the ability of genetic improvement through the correction of altered (mutated) genes or site-specific modifications that target therapeutic treatment. This therapy became possible through the advances of genetics and bioengineering that enabled manipulating vectors for delivery of extrachromosomal material to target cells. One of the main focuses of this technique is the optimization of delivery vehicles (vectors) that are mostly plasmids, nanostructured or viruses. The viruses are more often investigated due to their excellence of invading cells and inserting their genetic material. However, there is great concern regarding exacerbated immune responses and genome manipulation, especially in germ line cells. In vivo studies in in somatic cell showed satisfactory results with approved protocols in clinical trials. These trials have been conducted in the United States, Europe, Australia and China. Recent biotechnological advances, such as induced pluripotent stem cells in patients with liver diseases, chimeric antigen receptor T-cell immunotherapy, and genomic editing by CRISPR/Cas9, are addressed in this review.

RESUMO A habilidade de fazer modificações pontuais no genoma humano tem sido o objetivo da medicina desde o conhecimento do DNA como unidade básica da hereditariedade. Entende-se terapia gênica como a capacidade do melhoramento genético por meio da correção de genes alterados (mutados) ou modificações sítio-específicas, que tenham como alvo o tratamento terapêutico. Este tipo de procedimento tornou-se possível por conta dos avanços da genética e da bioengenharia, que permitiram a manipulação de vetores para a entrega do material extracromossomal em células-alvo. Um dos principais focos desta técnica é a otimização dos veículos de entrega (vetores) que, em sua maioria, são plasmídeos, nanoestruturados ou vírus − sendo estes últimos os mais estudados, devido à sua excelência em invadir as células e inserir seu material genético. No entanto, existe grande preocupação referente às respostas imunes exacerbadas e à manipulação do genoma, principalmente em linhagens germinativas. Estudos em células somáticas in vivo apresentaram resultados satisfatórios, e já existem protocolos aprovados para uso clínico. Os principais trials têm sido conduzidos nos Estados Unidos, Europa, Austrália e China. Recentes avanços biotecnológicos empregados para o aprimoramento da terapia gênica, como células-tronco pluripotentes induzidas em pacientes portadores de doenças hepáticas, imunoterapia com células T do receptor do antígeno quimera e edição genômica pelos sistema CRISPR/Cas9, são abordados nesta revisão.

Construction of Smac siRNA lentiviral vector and its silencing effect on Smac gene in human LECs / 中华实验眼科杂志

Background Research comfirmed that second mitochondrial activator of caspase (Smac) is a promoting tumor cell apoptosis protein.Our previous study showed that the expression level of Smac in LECs is obviously higher in cataract than that in normal eyes.We assumed that silencing Smac gene in LECs can inhibit the apoptosis of LECs.The way to transfect Smac siRNA into LECs is a key step.Objective This study was to construct siRNA lentiviral vector of Smac and identify its silencing efficiency in human lens epithelial B3 cell line (HLE-B3) and establish low-expressed Smac HLE-B3 line.Methods Based on the genebank and our previous study,siRNA sequence of Smac was designed and composed.The synthetic double-stranded DNA was linked to the lentiviral vector GVll8 by T4 DNA ligase and then transformed DH5α competent cells.The plasmids were transformed into the DH5α competent cells.Recombinant colonies were screened by PCR and sequenced.Recombinant plasmids and two other auxiliary plasmids were used to infect 293T cells.Cell culture supernatant was collected for the measurement of viral titer.Recombinant lentiviral vector was used to infect HLE-B3 cells to calculate the viral multiplicity of infection (MOI) under the fluorescence microscope.Transfection efficiency was examined by calculating the GFP-positive cells.HLE-B3 cells were divided into negative control group,siRNA plasmid tranfected group and GV118-Smac-siRNA1 tranfected group.The relative expression levels of Smac mRNA in the cells were detected and compared among the three groups by real-time fluorescent quantitative PCR.Results GV118-Smac-siRNA was successfully constructed with the positive colonies 340 bp and blank vector colonies 299 bp,and viral titer was 3.0× 108 TU/ml.At a MOI of 100,the infecting efficiency of the vector on HLE-B3 cells was about 82％ and the cytotoxicity was low.The relative expression levels of Smac mRNA were (101.290±8.349)％,(92.330±6.320)％ and (32.540±4.221)％ in the negative control group,siRNA plasmid tranfected group and GV118-Smac-siRNA1 tranfected group,respectively,showing a significant difference among the three groups(F =32.871,P＜0.01),and the relative expression level of Smac mRNA was significantly lower in the GV118-Smac-siRNA1 tranfected group than that in the negative control group (P =0.000).However,no significant difference was found in the Smac mRNA expression between the blank plasmid group and the negative control group (P=0.535).Conclusions GV118-Smac-siRNA lentiviral vector is successfully constructed.Smac-siRNA can effectively inhibit the expression of Smac mRNA in human LECs.

The construction of human prostate specific homeobox gene NKX3.1 eukaryotic expression vector and preliminary study on the biological function of NKX3.1 / 中国医师杂志

Objective To investigate the basic biological functions of NKX3.1 with construction of a series of eukaryotic expression vector of NKX3.1 to study its effects on cell cycle and apoptosis.Methods The full-length reading frames (ORF) of NKX3.1 was amplified from normal human prostate tissue with polymerase chain reaction (PCR),the ORF was cloned into T vector,and then NKX3.1 was cloned into eukaryotic expression vector pcDNA3.1 (+) and pcDNA3.1 (-) with this T vector as template.NKX3.1 in pEGFP C1 was transfected into PC3 cell line,and C1 pEFP was taken as control to observe the localization of NKX3.1 in cells.At the same time,NKX3.1 in pcDNA3.1 and pcDNA3.1 were transfected into PC3 cells and the cells were collected after 24 ～48 hours to detect cell cycle and apoptosis by flow cytometry.Results The eukaryotic expression vector of NKX3.1 was constructed successfully.Fluorescent protein was ocated in the nucleus after transfection of NKX3.1 in pEGFP C1,but pEGP C1 was both expressed in nucleus and cytoplasm.There was significant effect on the early apoptosis (P＜0.01),but no effect on the cell cycle (P ＞ 0.05) after transfection of NKX3.1 compared to PC3 cell-transfected with empty vector of pcDNA3.1.Conclusions The result data suggest that NKX3.1 be nuclear protein and have effect on the early apoptosis of PC3 cell line based on the construction of eukaryotic expression vector.

Construction of human microRNA-21 eukaryotic overexpression vector and its up-regulation of c-myc gene expression in HepG2 .2 .15 cells / 重庆医学

Objective To construct the miRNA‐21 eukaryotic overexpression vector pmR‐21 and to explore its regulation effect on the expression of c‐myc gene in HepG2 .2 .15 cells .Methods The miRNA‐21 precursor gene fragment pre‐miRNA‐21 was amplified by PCR ,then connected to the pmR‐mCherry plasmid vector after double enzyme digestion ,the accuracy of the recombi‐nant vector was verified by double enzyme digestion and sequencing ;then the recombinant vector was transfected into HepG2 .2 .15 cells ,the fluorescent protein expression was observed under the fluorescence microscopy at 24 h and the transfection efficiency was detected by flow cytometry ;the expression of miRNA‐21 was evaluated by real‐time quantitative PCR;at 72 h after transfection ,the expression levels of c‐myc gene were detected by RT‐PCR and Western blot ;CCK‐8 was used to detect the cell proliferation in each group .Results The double enzyme digestion and Western blot verified that the target gene fragment was inserted into the pmR‐mCherry vector;at 24 h after transfection ,intracellular strong fluorescence was seen ,the transfection efficiency was higher than 50% ;miRNA‐21 expression level of the pmR‐21 recombinant vector group was significantly increased;c‐myc gene expression was increased in the pmR‐21 recombinant vector group at 72 h after transfection ,the cell proliferation in the pmR‐21 recombinant group was faster than that in the control group(P<0 .05) .Conclusion The pmR‐21 eukaryotic overexpression vector is successfully con‐structed ,this recombinant vector can express miRNA‐21 stably ;miRNA‐21 can up‐regulate c‐myc gene expression ,c‐myc gene is one of miR‐21′s targets for playing a cancer‐promoting action .

Status analysis of gene therapy in osteosarcoma / 国际肿瘤学杂志

At present,the medical profession generally acknowledged the best way to treat osteosarcoma is gene therapy,which includes tumor suppressor gene therapy,antisense gene therapy,suicide gene therapy,immune gene therapy,combined gene therapy,etc.But no matter what kind of gene therapy is that the gene must have a safe carrier.Gene therapy has made a breakthrough in osteosarcoma recently.On the basis of widespread use,we should emphasize the importance of gene vectors.

pIRES2-EGFP-NT3 transfected mouse cochlea fibroblast by lipofectamineTM 2000 / 中国医师杂志

Objective To detect the transfection of pIRES 2-EGFP-NT3 in mouse cochlea fibroblast by cationic liposome . Methods After pIRES2-EGFP-NT3 had been abstracted successfully , it was transfected into mouse cochlea fibroblast by lipofectami-neTM2000.Twenty four hours later, the efficiency of the transfection was analyzed by confocal microscope .Results The pIRES2-EG-FP-NT3 was effectively transfected into mouse cochlea fibroblast by cationic liposome .The transfected fibroblasts displaying green fluo-rescence were observed under fluorescence microscope .Conclusions The effective transfection of pIRES 2-EGFP-NT3 into mouse cochlea fibroblast by lipofectamine TM 2000 laid the basis for the following experiments , such as NT3 gene transfection in deaf or normal cochlea and so on .

Construction and Application of Lentiviral Vectors Expressing microRNA-203 Tough Decoy / 天津医药

Objective To establish method of constructing lentiviral vectors to express microRNA (miRNA) ''tough decoy''(TuD)and to detect the effects of the TuD on cellular endogenous miRNA level and cellular phenotypes. Methods Two-step cloning strategy was utilized to first generate a universal miRNA TuD frame vector,followed by con-structing the TuD expression vector specially targeting miR-203. The package of the recombinant lentivirus was per-formed in 293T cells. Then the rat bone marrow mesenchymal stem cells(BM-MSCs)were infected by the miR-203 TuD expression lentivirus. The pSIH1-H1-copGFP vector was also packaged and the BM-MSCs infected by this lentivirus were served as control. Endogenous miR-203 level in BM-MSCs was measured by quantitative RT-PCR,and cellular vi-ability and apoptosis were detected by CCK-8 test and Annexin V-PI staining respectively. Results The miR-203 TuD expression vector was successfully constructed and inserted sequence was validated. At the 3rd,6th and 9th days after in-fected by the miR-203 TuD expression lentivirus,rat BM-MSCs exhibited a repressed endogenous miR-203 level. The miR-203 TuD also promoted viability and inhibited apoptosis of BM-MSCs. All these differences between miR-203 TuD group and control group were statistically significant. Conclusion The two-step cloning method for the construction of miRNA TuD expression vector is simple and efficient. The miRNA TuD can effectively suppress the level of the target miRNA and affect cellular phenotypes.

Effect of lentiviral vector-mediated GDNF up-regulation on neuropathic pain of chronic constriction injury rats / 中国医师杂志

Objective To investigate the effect of intrathecal injection of lentiviral vector-mediated up-regulation of glial cell line-derived neurotrophicfactor (GDNF) on neuropathic pain of chronic constriction injury (CCI) rats.Methods The CCI model was prepared by ligating the sciatic nerve of Sprague-Dawley (SD) rats.Seven days after CCI modeling,a single intrathecal injection of lentiviral vectors (LV)-GDNF was given.Before CCI and 3,5,7,14,and 21 days after CCI modeling,the mechanical pain threshold was tested in rats,and 21 days after surgery,Western blot was used to detect the expression of GDNF protein.Results On 21 days after CCI modeling,GDNF expression was reduced compared to sham group.After intrathecal injection of LV-GDNF,GDNF expression was up-regulated in the spinal cord,and CCI-induced mechanical hyperalgesia in rats was alleviated.Conclusions Intrathecal injection LV-GDNF can up-regulate the expression of GDNF and alleviate neuropathic pain in CCI rats.

Inhibition of tumor growth by intramuscular administration of the canstatin gene delivered by electroporation / 中国医师杂志

Objective To construct human canstatin gene eukaryotic expression vector and investigate the therapeutic effect of intramuscular canstatin gene delivered by electroporation on tumor growth.Methods Canstatin cDNA was amplified from total RNA extracted from fresh fetal liver by reversing transcription polymerase chain reaction (RT-PCR).The canstatin cDNA fragment was in serted into pEGFP-N1 eukaryotic expression vector.The recombination plasmid was delivered to the quadriceps of the mice with Lewis lung carcinomas by electroporation intramuscular.Fluorescence intension measured by fluorescence microscope,reverse-PCR assay,and immunohistochemistry assay were performed to detect the expression of canstatin gene in the muscle and in circulation.The tumor weight and volume were used to detect the biological effects of canstatin gene delivery.Results Recombinant eukaryotic expression vector of recombinant human canstatin was successfully constructed.The canstatin mRNA was significantly increased in the skeletal muscle and intramuscular delivery of canatatin gene by electroporation acquired the expression of enhanced green fluorescent protein (EGFP)/canstatin protein in the circulation and significantly inhibited tumor growth.The percent of the inhibition of tumor weight was 57.7 ％.Conclusions Electroporation mediated gene transfer efficiency in skeletal muscle was compared to simple plasmid injection and lasted for a long time.It was an efficient and safe,convenient and economic,gene transfer methods and might have certain clinical application value.Electroporation mediated canstatin gene transfer in skeletal muscle had obvious inhibitory effect on Lewis lung cancer in mice subcutaneous xenograft tumor growth.

Development of porcine respiratory and reproductive syndrome virus replicon vector for foot-and-mouth disease vaccine

PURPOSE: Foot-and-mouth disease (FMD) is an economically important global animal disease. To control FMD virus (FMDV) outbreaks, a lot of different novel approaches have been attempted. In this study, we proposed a novel porcine reproductive and respiratory syndrome virus (PRRSV) as a replicon vector to express FMDV structural protein. MATERIALS AND METHODS: PRRSV infectious clone (PRRSVK418DM) was used to develop an expression vector through the reverse genetic manipulation of PRRSV; FMDVP12A3C gene of serotype O was synthesized and used for an antigen. MARC-145 cells (African green monkey kidney epithelial cell line) were used for electroporation mediated transfection. The transfection or the expression of P12A3C and N protein of PRRSV was analyzed by either replicon containing PRRSV alone or by co-infection of helper PRRSV. RESULTS: We constructed PRRSVK418DM replicon vector containing FMDVP12A3C, and genome sequences were confirmed by subsequent sequence analysis. In vitro expression of P12A3C and PRRSV N protein was confirmed by immunofluorescence antibody assay using antibodies specific for PRRSV N protein (anti-PRRSV N MAb), FMDV-VP1 (anti-VP1 MAb). CONCLUSION: The results indicate that PRRSV replicon vector can be a promising novel vector system to control FMDV and useful for vaccine development in the future.

Transfection of Lentivirus Recombined with Marker Gene into Human Umbilical Cord Wharton’s Jelly-Derived Mesenchymal Stem Cells / 天津医药

Objective To investigate the optimal condition of lentivirus,which was recombined with marker gene of enhanced green fluorescent protein (Lentivirus-EGFP) transfect human umbilical cord wharton’s jelly-derived mesenchy-mal stem cells (HUWMSCs) and the effect of transfection on the proliferation in HUWMSCs. Methods HUWMSCs were transfected with EGFP by lentivirus vector in vitro via different multiplicity of transfection (MOI) in four different transfec-tion methods (A, B, C and D). The fluorescence expression and the transfection efficiency in different methods were analyzed by both fluorescent microscope and flow cytometry. The proliferation rates of infected HUWMSCs was evaluated by MTT method. Results The transfection efficiency was 10.6%-87.3%after 4 days in all experimental groups, which showed the dose-effect relationship with MOI. Polybrene (5 mg/L) could significantly increase the transfection efficiency (P＜0.05). Re-sults of MTT assay showed that there were significant differences in the proliferation rates of infected HUWMSCs between different transfection methods (P ＜ 0.001). There was better cell proliferation in method A (MOI=10) group and method B (MOI=10) group than that of other groups. Conclusion Method B (MOI=10) is the optimal transfection method in this exper-iment. HUWMSCs could be transfected by lentivirus-EGFP with high efficiency and could stably express transfected gene within 2 weeks, which is a safe and effective gene transfer vector.

Construction of eukaryotic expression vector of iASPP and its identification of biological function / 重庆医学

Objective Construct the eukaryotic expression vector of inhibitory member of the ASPP family (iASPP) and trans-fect it into colon carcinoma cell lines SW480 and Lovo by liposome .Then observe the expression of iASPP and detect the cell apop-tosis by flow cytometry .Methods The amplified PCR product was digested and inserted into pMD19-T simple vector and sub-cloned into eukaryotic expression vector pcDNA3 .1(+ ) .The recombinant eukaryotic expression plasmid pcDNA3 .1(+ )-iASPP was transfected into colon carcinoma cell lines SW480 and Lovo by liposome ,the iASPP expression was analyzed by RT-PCR .The cell apoptosis was detected by FCM .Results The eukaryotic expression plasmid pcDNA3 .1(+ )-iASPP was constructed success-fully ,the gene squence of iASPP was consistent with that reported (gi 60457962) in GenBank .The mRNA expression levels of iASPP gene of SW480 and Lovo cell lines which transfect the positive plasmid were increased ,and the cell apoptosis rates were de-creased .Conclusion We successfully constructed the recombinant expression plasmid pcDNA 3 .1(+ )-iASPP ,and the plasmid were successfully expressed in colon carcinoma cell lines SW 480 and Lovo ,the cell apoptosis rates of those cell lines were decreased .These facts indicated that reducing the high expression of iASPP may be a new strategy to renew the abilities of P 53 tumor suppressor .

Intratumoral enrichment of gene delivered by oral administration of recombinant attenuated Salmonella typhimurium in tumor-bearing nude mice / 第二军医大学学报

Objective To observe the distribution and expression of exogenous gene delivered by oral administration of recombinant attenuated Salmonella typhimurium in nude mouse model carrying human saliva gland adenoid cystic carcinoma. Methods The attenuated Salmonella typhimurium of green fluorescent protein (GFP) gene expression was constructed by transfecting prokaryotic and eukaryotic expression plasmid vector into the attenuated Salmonella typhimurium SL7207, and the recombinant attenuated Salmonella typhimurium SL7207-pUC-GFP for prokaryotic expression and SL7207-pEGFP-Nl for eukaryotic expression were obtained. The expression stability of GFP gene in the prokaryotic SL7207-pUC-GFP was tested for 10 times in vitro. The nude mouse models carrying human saliva gland adenoid cystic carcinoma were orally administered with prokaryotic SL7207-pUC-GFP (0. 1 mL,l×109 cfu/mL), and then the distribution of the attenuated Salmonella typhimurium in the liver, spleen and tumor tissues were assessed by observing GFP expression in SL7207-pUC-GFP clones cultured out of the tissues homogenate at 24 h, 48 h, 5 d, 10 d, 20 d, and 30 d after oral administration. The nude mouse models were also orally administered with eukaryotic SL7207-pEGFP-Nl, and the expression of GFP in the liver, spleen and tumor tissues was observed in frozen tissue sections 5 d later under fluorescence microscope. Results The expression of GFP harbored by the recombinant attenuated Salmonella typhimurium SL7207-pUC-GFP was not reduced or lost after 10 times of in vitro passaging. After the oral administration of prokaryotic SL7207-pUC-GFP, the attenuated Salmonella typhimurium could survive in the liver, spleen and tumor tissues for a long time, with the clone numbers in tumor tissues being significantly higher than those in the liver and spleen tissues at all the time points (P<0. 05). After oral administration of eukaryotic SL7207-pEGFP-Nl, the expression of GFP was higher in tumor tissues than in the liver and spleen tissues. Conclusion The recombinant attenuated Salmonella typhimurium can be enriched in human saliva gland adenoid cystic carcinoma cells and deliver the harbored exogenous gene into the tumor cells for expression, showing a double advantage for gene therapy.

Transfection of Lentivirus Vector Containing Apelin Gene into Umbilical Cord Mesenchymal Stem Cells in Vitro / 天津医药

Objective To construct a recombinant plasmid pUbi-apelin-FLAG-pSV40-EGFP and package with lentivirus to co-express enhanced green fluorescent protein (EGFP) and apelin, and to investigate optimal multiple of infec-tion (MOI) to transfect human umbilical cord mesenchymal stem cells (hUCMSCs) and expression of target gene. Methods The apelin gene was chemically synthesized and amplified by polymerase chain reaction (PCR), and which was inserted into linear plasmid vector. The gene fragment and linear plasmid vector were connected by In-Fusion technology after enzyme di-gestion and transformed into competent DH5αcells. The positive clones of lentiviral expression vector were obtained after screening and followed by sequencing. The lentiviral vector was used to transfect 293T cells and package virus, and then the virus titers were determined. HUCMSCs were transfected with lentivirus vector in vitro via different values of MOI. The trans-fection efficiency was obtained according to the optimal MOI. The expression of target gene was detected by RT-PCR and Western blot assay. Results A 284 bp target gene fragment with the restriction sites was obtained by PCR and connected to the lentiviral vector. The positive clones of lentiviral expression vector were corresponded to the expected result. The lentivi-ral particles were successfully packaged. HUCMSCs could be transfected by the lentivirus vector with high efficiency. The mRNA and protein levels of target gene were stably up-regulated within 2 weeks. Conclusion The lentivirus vector pUbi-apelin-FLAG-pSV40-EGFP can transfect apelin gene into hUCMSCs with high efficiency. The infected cells can express high levels of apelin gene in two weeks.

Prokryotic expression of CFP-10 protein of Mycobacterium tuberculosis / 中国医师杂志

Objective To construct recombinant plasmid containing CFP-10 gene of Mycobacterium tuberculosis(MTB).Methods The gene fragment of CFP-10 was amplified by PCR from Mycobacterium tuberculosis H37Rv genomic DNA and cloned to pET-32a(+) vector.The recombinant plasmid pET-32a-CFP-10 was transformed into E.coli BL21 (DE3) and induced by IPTG.Results CFP-10 gene fragment was amplified from genomic DNA of Mycobacterium tuberculosis H37Rv strain,and thepET-32a(+) prokaryotic recombinant plasmid was constructed successfully.The recombinant protein was expressed with the induction of IPTG.Conclusions The prokaryotic expression vector for CFP-10 was successfully constructed and the recombinant protein was highly expressed in E.coli BL21 (DE3),which lays a foundation for its subsequent immunological function study.

Desarrollo de u poxvirus recombinante que exprsa la glicoproteína d del herpeesvirus bovino-1 / Development of a Recombinant Poxvirus Expressing Bovine Herpesvirus-1 Glycoprotein D

El herpesvirus bovino-1 (BHV-1) es un virus de genoma DNA perteneciente a la familia Herpesviridae, el cual afecta al bovino en el que provoca un amplio espectro de manifestaciones clínicas y pérdidas económicas. El principal componente inmunogénico de su envoltura es la glicoproteína D (gD), la cual ha sido caracterizada y utilizada como inmunógeno en distintos sistemas de expresión. El objetivo de este trabajo fue generar un poxvirus recombinante (Raccoonpox [RCN]) que expresara una versión truncada de la gD del BHV-1 para ser usado como inmunógeno. Para ello, se amplificó el gen que codifica para la versión truncada de la gD, la cual se clonó en el plásmido de transferencia pTK/ IRES/tpa que posee sitios de homología a la timidinakinasa del poxvirus, un sitio interno de entrada al ribosoma (IRES) y una señal secretoria (tPA), generando el constructo pTK/gD/IRES/tpa. Para generar el RCN recombinante, se tomaron células BSC-1, se infectaron con una cepa Silvestre del RCN (CDC/V71-I-85A) a un índice de multiplicidad de infección de 0,05 y se transfectaron con el constructo pTK/gD/IRES/tpa; generándose diferentes poblaciones virales con y sin el gen de interés. Para seleccionar los virus recombinantes que expresaban el gen de interés, se realizó una selección de recombinantes negativos para timidina kinasa y positivos para la gD por tres rondas de purificación de placas en monocapas de células RAT-2 las cuales son mutantes para timidina kinasa y en presencia de bromodeoxiuridina. Los virus recombinantes se confirmaron por PCR y secuenciación de nucleótidos y se denominaron RCN-gD.

Bovine Herpesvirus-1 is a DNA virus belonging to the family Herpesviridae, which affects cattle, causing a wide spectrum of clinical manifestations and economic losses. The main immunogenic component is its envelope glycoprotein D (gD), which has been characterized and used as immunogen in different expression systems. The aim of this work was to generate a recombinant poxvirus (Raccoonpox [RCN]) expressing a truncated version of BHV-1 gD to be used as a vaccine. To do this, it was amplified the gene for a truncated version of gD which subsequently was cloned in transfer plasmid PTK/IRES/tpa which has homology to sites of poxvirus thymidine kinase, an internal site of ribosome entry (IRES) and a secretory signal (tPA), generating the construct PTK/gD/IRES/tpa. To generate the recombinant RCN, we took BSC-1 cells and we infected with a wild type RCN (CDC/V71-I-85A) at a multiplicity of infection of 0.05, then cells were transfected with the construct PTK/gD/IRES/tpa, generating different viral populations with and without the gene of interest. To select recombinant viruses expressing the gene of interest, we performed a selection of recombinant thymidine kinase negative and positive for gD by three rounds of plaque purification on RAT-2 cells monolayers which are thymidine kinase null and using bromodeoxyuridine. Recombinant viruses were recovered and confirmed by PCR and nucleotide sequencing and so called RCN-gD.