Construction And Identification Of A Type 5 Adenovirus Vector Carrying Human Interleukin-29 Gene

Objective:Cancer is caused by the abnormal proliferation of cells under the combined actions of environmental factors,hereditary factors and other factors,and the molecular basis is the imbalance of gene expression regulation system which is caused by DNA mutation.In addition to surgery,chemotherapy and radiotherapy,gene therapy and immunotherapy are also developing rapidly.It is difficult to effectively lengthen the lifetime of cancer patients with single therapeutic scheme,so the combination of multiple therapeutic schemes becomes a common treatment principle.Esophageal cancer is one of the common gastrointestinal malignancies.The morbidity and mortality rates of our country leap into the front ranks of the world,and more than 200,000 people deaths every year.The pathogenesis of esophageal cancer is complex,and a variety of regulatory factors are involved in cell carcinogenesis.For patients whose clinical stages are early,radical operation is the preferred therapeutic method.But in more than 90%of all patients with esophageal cancer,the cancer is detected in an advanced stage,they already can't tolerate surgery,therefore,gene therapy and immunotherapy provide a feasible option for combined chemotherapy or radiotherapy.In recent years,with the development of molecular biology,gene therapy has been applied to clinical therapy gradually.Whether gene therapy is effective or not largely depends on the effective delivery of target gene,and gene therapy needs effective vectors.At present,the vectors such as adenovirus,herpes virus,retrovirus and vaccine virus are the most applicable viral vectors,which have been widely used in animal or clinical studies of gene therapy.Because adenovirus vector has the advantages such as expression the target gene,high security and so on,in this study,we used adenovirus vector as the delivery vector of IL-29 gene.Human interleukin-29?IL-29?locates on the long arm of chromosome 19?19q13.13?,the total length of its mRNA is 856 bp,the open reading frame of is 603 bp,and it is a member of the IL-10 cytokine superfamily.The biological functions of IL-29 mainly include anti-virus,anti-proliferation,immunoregulation and so on.However,cytokines have some disadvantages such as small molecular weight and short half-life.This study aims to recombine IL-29 gene through molecular cloning technique,construct pAd5-IL-29,prepare Ad5-IL-29,and explore the anti-proliferation and its mechanism of Ad5-IL-29 in esophageal cancer cell line.This study will provide experimental basis and theoretical basis for the pre-clinical study of Ad5-IL-29-mediated gene therapy in esophageal cancer.Methods:1 Construction of pAd5-IL-29The IL-29 gene fragment was amplified by RT-PCR from the total RNA extracted from HaCaT cells stimulated by poly I:C,then purified and extracted.The connection products that IL-29 gene fragment and p CR2.1 was identified by enzyme digestion and gene sequencing.The IL-29 gene fragment was digested with Kpn I and Not I in turn from pC R2.1-IL-29 and was constructed into pShuttle2 that was digested with K pn I and Not I,then pShuttle2-IL-29 was extracted after transformation and amplification.pShuttle2-IL-29 was identified by enzyme digestion af ter digesting with Kpn I and Not I respectively.pShuttle2-LacZ,pShuttl e2-IL-29 and pAd5 were digested with I-Ceu I and PI-Sce I,the digesti on products of pShuttle2-LacZ and pShuttle2-IL-29 connected with the d igestion products of pAd5 respectively.pAd5-LacZ and pAd5-IL-29 were extracted after transformation and amplification,then the connection pro ducts that were digested by Swa I were identified by enzyme digestion after digesting with Hind III.2 Transfection of pAd5-IL-29 and preparation of Ad5-IL-29The adenovirus vectors that carrying LacZ and IL-29 encoding sequences were digested with PacI,the digestion products were transfected into 293 cells by lipofection.Ad5-LacZ and Ad5-IL-29 were extracted after cytopathic effect,then both infected 293 cells to amplify recombinant adenovirus enough.Ad5-LacZ and Ad5-IL-29 were purified with Adeno-X^TM virus purification kits?BD Biosciences,Clontech?,checked the titer by tissue culture infective dose,and calculated the titer with the method of KARBER:T=10^1+d?s-0.5?.3 Identification the expression of IL-29 with Western blot assayEsophageal cell YES2 was treated with Ad5-LacZ and Ad5-IL-29 at MOI of 3000,supernatant was harvested and the proteins were denatured through heating.Then the proteins were subjected to SDS-PAGE and probed with monoclonal antibody anti-hIL-29?R&D Systems?and the corresponding second antibody.The membranes were developed with the ECL system.4 Cell proliferation assayEsophageal cancer cells including TE1,TE2,TE10,YES2,YES5 and YES6 were infected with Ad5-LacZ and Ad5-IL-29 at MOI of 0,30,100,300,1000 or 3000,in three independent experiments.The proliferation rate was detected with a CCK-8 reagent,and the results were determined with the absorbance at 450 nm.The proliferation rate was calculated on the basis of the absorbance without any treatment.5 Cell cycle analysis by flow cytometryEsophageal cancer cell YES2 was infected with Ad5-LacZ and Ad5-IL-29 at MOI of 3000,in three independent experiments,cells were stained with a reagent of propidium iodide,and cell cycle distributions were then analyzed by flow cytometry after infection for 24 h and 48 h.6 Statistical analyses were performed by SPSS 21.0 software.Each set of data was tested for normality and homogeneity of variance.Data were expressed as Mean±SD.If multiple sets of variables were consistent with homogeneity of variance,the single factor analysis of variance?one-way ANOVA?was used to compare multi-group variables,otherwise,was analyzed by nonparametric test.The comparison among groups was performed with the method of least significant differences?Student-Newman-Keuls,SNK?.Two-sided P values were used and P?0.05 indicated that the differences are statistical significance.For each protocol,at least three independent experiments were performed.Results:1 Construction pAd5-LacZ and pAd5-IL-29IL-29 gene was cloned into pCR2.1 and pCR2.1-IL-29 was constructed,IL-29 gene sequence was identified by gene sequencing,which was consistent with the sequence published by GenBank?IL-29:AY129150?.IL-29 gene fragment acquired through digesting pCR2.1-IL-29 with Kpn I and Not I in turn,was subcloned into pShuttle2 which was digested with the same endonucleases.pAd5-LacZ and pAd5-IL-29 were constructed through connected the digestion products of pShuttle2-LacZ or pShuttle2-IL-29 and pAd5,which were digested with I-Ceu I and PI-Sce I.2 Preparation of Ad5-LacZ and Ad5-IL-29 and identification the expression of IL-29pAd5-LacZ or pAd5-IL-29 was transfected into 293 cells with lipofection.After infection in 293 cells,the titer of Ad5-LacZ and Ad5-IL-29 were checked after amplification and purification.By the means of Western blot,the experimental group that Ad5-IL-29 infected YES2 showed specific protein which was detected that molecular weight of IL-29 was about 23 kDa,but that of the control group was not detected.The data showed that pAd5-IL-29 was constructed successfully,and IL-29 was expressed in YES2 infected by Ad5-IL-29.3 Preliminary study of the anti-proliferative mechanism mediated by Ad5-IL-293.1 The proliferative inhibition of Ad5-IL-29 in esophageal cancer cellsCompared with the control group Ad5-LacZ,with the increase of MOI,the proliferation rates induced by Ad5-IL-29 in all the cells were decreased in different degrees,but in the cells of TE1,TE10 and YES6,the differences were no statistical significance?P>0.05?,in the cells of TE2,YES2 and YES5,the differences were statistical significance?P<0.05?.Compared with Ad5-LacZ,Ad5-IL-29 could induced prolifation inhi bition in the cells of TE2,YES2 and YES5,but not in the cells of TE1,TE10 and YES6.3.2 The cell cycle change induced by Ad5-IL-29 in YES2 cellsYES2,the sensitive cell,was infected with Ad5-LacZ and Ad5-IL-29 and cultured for 24h and 48 h,the cell cycle distributions were detec ted with flow cytometry.The results showed that sub-G1 fraction and S fraction were increased in YES2 as early as 48 h after the Ad5-IL-29infection,compared with Ad5-LacZ?P<0.05?.Conclusions:1 pAd5-IL-29 was constructed,and the expression of IL-29 was mediated in YES2 which was infected by Ad5-IL-29.2 Ad5-IL-29 could inhibite the proliferation of esophageal cancer cells,but its susceptibility was different among the esophageal cancer cells.The mechanisms of the anti-proliferation of Ad5-IL-29 in the sensitive cells may be cell apoptosis and cell cycle arrest in the S-phase.