Cloning, Prokaryotic Expression, And Identification Transcription Profile Of The Novel Brain Biomarker Vsnl1 Gene In Rat Ischemic Stroke Models

Cerebral ischemic stroke(IS) remains one of the leading disorders with high mortality and long-term neurological disability throughout the world.About 30%of patients die of stroke within a year.By 2020,stroke mortality will have almost doubled mainly because of an increase in the proportion of older people.Sometimes,it is still difficult to make diagnosis especially in the very early stage even through CT and MRI.A lot of molecular biomarkers specific for brain tissues have ever been demonstrated to participate in the pathogenesis of IS,the application of which will be helpful for earlier diagnosis of suspected stroke.As reported by the Ladenson group of Washington University in June 2006,the visinin-like protein 1(VILIP-1) was screened out and identified to be abundant and specific in brain through gene array,bioinformatics and Western blot assays.Also,they found that VILIP-1 level increased in IS patients and animal models.So,it seems that VILIP-1 has been a prospective biomarker for ischemic injury in brain.The VILIP-1 is encoded by vsnl1 gene with 576 bp in length,the sequence of which remain same homologous among Human,Mouse and Rat according to GenBank data. VILIP-1 has widespread distributions and high expression levels in many regions containing basal ganglia,procerebrum,cerebellum and cerebrum,and in most neuronal cells of brain.VILIP-1 is a member of VILIP subfamily,which belongs to the superfamily of neuronal calcium sensor(NCS) proteins.The constitutive expression of VILIP-1 in normal brain tissues has been relatively clarified.However,transcription profile of vsnl1 remains unclear especially in brain suffered from ischemic injury.In the present study,we have cloned vsnl1 gene and made prokaryotic expression of VILIP-1,and made rat models of IS,in which the transcription profiling of vsnl1 has then been identified.1.Cloning and prokaryotic expression of vsnl1 geneFirstly,PCR primers was designed by PrimerSelect software used to amplify vsnl1. Then,total RNA was extracted from healthy BALB/c mice,purified by BIOZOL reagent, digested with RNase-free DNase I,and reversely transcribed to cDNA,which was used as the template of PCR.The target fragment was inserted into pMD18-T vector,and nucleotide sequence was verified by sequencing.The vsnl1 sequence was subscribed to GenBank and subjected to online BLAST analysis.Then,recombinant prokaryotic expression plasmid pROEX-v containing full-length vsnl1 was constructed by subcloning technique,and transformed into E.coli DH5a,which was then induced to produce VILIP-1 by IPTG.Results were shown as follows.(1) The target product of RT-PCR was of 576 bp, being the same to vsnl1.(2) We aligned the vsnl1 with previously published sequence (AY101375) by online BLAST tool and found two mutation sites consisting of,nt205(T-C) and nt314(T-A).The GenBank accession no.of our sequence is EU373446.(3) The 22-KDa VILIP-1 was successfully expressed by pROEX-v plasmid in E.coli and purified by Ni-NTA column,which were verified by SDS-PAGE.Unfortunately,the yield rate of purified protein was low.2.Performation of IS rat models and studies on vsnl1 transcription profilingFirstly,models of IS were made through permanent or transient occlusion of right middle cerebral artery(pMCAO,n=11;tMCAO,n=11;conrol,n=10) according to previously described methods with some modifications.Neurological deficits of models were evaluated at 24 h after operation,brain sections of which were then sliced and stained with 2%TTC and hematoxylin-eosin(H.E),respectively.Secondly,the whole brain was removed after trans-cardial perfusion and total RNA was extracted,purified by BIOZOL reagent,digested with RNase-free DNase I,and reversely transcribed to cDNA. Subsequently,semi-quantitative PCR and agarose gel electrophoresis was made and target bands were determined by Quantity One software.Then,ratios of transcript level of vsnl1 mRNA relative toβ-αctin mRNA were statistically analyzed.Lastly,real-time PCR primers were designed by DNAMAN and PrimerSelect soflwares used to identify vsnl1 transcription level in MCAO brain tissues.Experimental results were displayed as follows.(1) MCAO animals exhibited obvious neurological deficits at 24 h after operation,whereas sham-operated animals showed normal activities.Neurological severity scores in either pMCAO(2.64±1.03) or tMCAO (2.27±0.90) groups had significant difference with those in control(0) group(P＜0.05). Furthermore,it seems that tMCAO rats had longer survival time than pMCAO ones,which reminds us that rats after IS may gain certain survive-related profits from cerebral reperfusion.(2) Photographed TTC-stained pictures have exhibited dramatic infarctions in blood-supply areas of the right MCA,including cerebral cortex,corpus striatum and parts of hippocampus.Moreover,the microscopy after H.E.staining has showed not only series of cellular morphologies including edema,degeneration and necrosis,but many infarction or micro-infarction focuses.As controls,slices from healthy brain displayed normal morphologies both at the gross level and under a microscope.Thus,histopathological results have further confirmed the success of rat MCAO models.(3) The semi-quantitative PCR results demonstrated that the vsnl1 transcription levels were similar between the left and right brains of sham-operated rats.However,in pMCAO and tMCAO rats,the right (ischemia) lateral of brain showed lower vsnl1 transcript level than the left(non-ischemia). (4) The ratios of vsnl1 mRNA/β-actin mRNA in ischemia(right) and non-ischemia(left) lateral of brain tissues were 0.040±0.007(left,pMCAO),0.023±0.002(right,pMCAO), 0.049±0.011(left,tMCAO) and 0.030±0.002(right,tMCAO),respectively.In both pMCAO and tMCAO models,the vsnl1 transcript level in the ischemic brain was lower than that in the normal brain,although without statistical significance.But in brains of control animals,relative vsnl1 transcript of both laterals showed similar levels.Here it is postulated that the disturbance of calcium level may be responsible for the vsnl1 downtranscription in the ischemic brain of MCAO models.Because of complicated roles of VILIP-1 in pro-apoptosis and multiple signaling cascades,to identify the concrete mechanism still needs further explorations.