Construction And Identification Of A Recombinant Adenovirus Vector Containing Rat ZnT1Gene

Objective1. To construct a recombinant adenovirus with ZnT1(Zinc transporter1) by λphasge-site specific recombination system and using enhanced greenfluorescent protein (EGFP) as resport molecular,which named pAd-CMV-ZnT1-IRES-EGFP. Then the expression vector transferred into HEK293A cells to bepackage and called Ad-CMV-ZnT1-IRES-EGFP. At the same time to constructpAd-CMV-IRES-EGFP as negative control vector.2. To transfer gene recombinant adenovirus expression vector and negativecontrol vector into HEK293A cells with optimum multiplicity of infection (MOI)and study expression quantity of the two vectors. We laid a good foundation forthe ZnT1gene to express in spinal cord nerve cell and the relationship withBDNF/TrkB signal regulation channel and establish a new way in molecularlevel to treat with spinal cord injury(SCI).MethodsUsing chemosynthesis method to synthesize the interest gene Znt1-IRES--EGFP.Adding attB1,attB2recombination site to the interest gene both endsthought PCR method，then the later and pDONR221vector with attP1,attP2rec-onmbination site create into a entry vector pDown-Znt1-IRES-EGFP throughBP recombination. The entry vector with attL1,attL2recombination site and thetarget vector Ad/CMV/V5-DEST were recombined together to create theexpression vector pAd-CMV-ZnT1-IRES-EGFP under LR reaction. Enzyme digesting and sequencing appraisal to prove the expression vector，digestingwith PacⅠenzyme, and transferred into HEK293A cells to be packaged, thenget the maturated adenovirus. To extract recombinant adenovirus plasmid afteresotoxin elimination and to recycle large fragment after PacⅠ restrictionenzyme with phenol and chloroform method. The later was transferred intoHEK293A cells under the help of Lipofectamine2000. We observed the EGFP’sexpression to appraise whether or not the package is successful. Usingendpoint dilution method to detect the virus titre and at last construction ofrecombinant adenovirus expression vector namely pAd-CMV-ZnT1-IRES-EGFPis accomplished. The negative control vector Ad-CMV-IRES-EGFP isaccomplished too. The expression vector and negative control vector wereinfected HEK293A cell, finally,the interest protein’s expression is analyzedusing western blotting technology.Results1. Enzyme digesting and sequencing appraisal to prove the expression vectorhave recombined into target vector with right orientation. The recombinantadenovirus expression vector pAd-CMV-ZnT1-IRES-EGFP is set up, at thesame time the negative vector pAd-CMV-IRES-EGFP is set up too.2. It is observed by fluorescence inverted microscope that a great quantityexpression of EGFP and obvious cytopathic effect (CPE) can be obtained inHEK293A cells which pAd-CMV-ZnT1-IRES-EGFP and negative control vectorhave been transfer into HEK293A cells. Packaging of adenovirus is successfuland virus titre are2.5×108pfu/ml and1.6×108pfu/ml respectively.3. It is found that quantity of ZnT1proteinum expression is higher than anothergroup′s and there is significance statistics (P＜0.01) but there is no significancestatistics among the negative group and normal group (P＞0.1) by Western blotdecection after transfection into HEK293A cells. ConclusionsA recombinant adenovirus expression vector namely pAd-CMV-ZnT1--IRES-EGFP was constructed successfully and efficiently which laid a goodfoundation for the ZnT1gene to express in spinal cord nerve cell and therelationship with BDNF/TrkB signal regulation channel.