Clone And Expression Of Splice Variants Ofinterleukin7

Interleukin7（IL-7） is an important immune regulatory factor, and the main functionis to promote B cell and T cell growth and resistance to apoptosis. It is currently exploringthe use of IL-7on the related disease treatment. Splice variants of IL-7derived from IL-7gene, but the primary structure of a protein and the IL-7are difference. At present, thestudy of its function is just beginning. To clone and select the splice variants in openreading frame of interleukin7from cancer cell lines （HepG2and HT29）. Splice variants ofIL-7were cloned into the pET21-b vector for protein expression in Escherichia coli strainBL21（DE3） and the Splice variants of protein would be identification. The study will forthe further investigating the splice variants of the biological activity to lay corporealfoundation.Methods:1. To clone IL-7and the splice variants of IL-7: Total RNA was extracted from HepG2and HT29with Trizol （Beyotime） as described by manufacturer, individual. IL-7and thesplice variants of IL-7genes were amplified by reverse-transcription chain reaction（RT-PCR） based on the nucleotide sequence of Homo sapiens Interleukin7（GenBankAccession No.NM₀00880） using specific primers, respectively. The resultant cDNAfragments were cloned into T vector to recombinant plasmids. The resultant plasmids weretransformed into E.coli DH5a cells to verify by PCR screening and sequencing.2. To construct recombinant IL-7and in the open reading frame of splice variants ofthe prokaryotic expression vector: IL-7and in its open reading frame splicing variantrecombinant plasmid were amplified by PCR using specific primers, which were digested with Bamh1and xhol restriction enzymes. The resultant DNA fragments were cloned intoT vector to recombinant plasmids. The resultant plasmids were transformed into E.coliDH5a cells to verify by PCR screening and restriction endonuclease analysis. The excisedproducts were subcloned into expression vector pET21b, previously digested with the samerestriction enzymes. The recombinant expression vector were transformed into E.coli DH5acells to verify by PCR screening and DNA sequencing.3. IL-7and in the open reading frame of splice variants of the prokaryotic expressionand identification: The recombinant expression vectors were finally transformed intoE.coli BL21（DE3） for protein expression. After induction by IPTG, the protein wasanalyzed by SDS-PAGE and determined by western blot.4. Analysis in the open reading frame of splice variants of IL-7: Using Internet toanalysis the protein of in the open reading frame of splice variants of IL-7.Results:1. To clone IL-7and the splice variants of IL-7: Total RNA of HepG2and HT29wereused as templates for cDNA synthesis. The DNA frame encoding was obtained by RT-PCRand the resultant cDNA fragments were cloned into pMD18-T vector. The recombinantplasmid were verified by PCR screening and DNA sequencing. The results confirmed thatthe sequences of IL-7was identifying to the sequences reported in GenBank. By analyzingthe splice variants, we find5of them in open reading frame of IL-7, including IL-7delta4with399bp、IL-7delta3/4with318bp、IL-7delta4/5with345bp、IL-7delta3/4/5with264bp. Also we find several splice variants was not in open reading frame of IL-7, includingIL-7delta2/4with343bp、IL-7delta2/3/4with268bp、IL-7delta2/3/5with181bp、IL-7delta2/4/5with289bp、IL-7delta2/3/4/5with208bp.2. To construct recombinant IL-7and in the open reading frame of splice variants ofthe prokaryotic expression vector: IL-7and in its open reading frame splicing variantrecombinant plasmid were used as templates for DNA synthesis. Then they were amplifiedby PCR using specific primers, which were digested with Bamh1and xhol restrictionenzymes. The purpose of using restriction fragment was subcloned into prokaryotic expression vector pET21b. The recombinant expression vectors were verified by PCRscreening and DNA sequencing. We got the recombinant expression vectors ofpET21b-IL-7、 pET21b-IL-7delta4、 pET21b-IL-7delta3/4、 pET21b-delta4/5、pET21b-IL-7delta3/4/5.3. IL-7and in the open reading frame of splice variants of the prokaryotic expressionand identification: The resultant recombinant expression vectors of pET21b-IL-7、pET21b-IL-7delta4、pET21b-IL-7delta3/4、pET21b-delta4/5、pET21b-IL-7delta3/4/5andpET21b were transformed into E.coli BL21（DE3）. After induction by IPTG, the protein wasanalyzed by SDS-PAGE and determined by western blot. And the proteins were targetproteins.4. Analysis in the open reading frame of splice variants of IL-7: we got somen dataabout in the open reading frame of splice variants of IL-7.Conclusions:Total RNA was extracted from HepG2and HT29with Trizol and the DNA frameencoding was obtained by RT-PCR.We obtained with GenBank published the sequencesidentical of IL-7. Also we got several splice variants of IL-7and five of them in IL-7’sopen reading frame. The splice variants were subcloned into prokaryotic expressionplasmid pET21b. And then we got the recombinant expression vectors of pET21b-IL-7、pET21b-IL-7delta4、pET21b-IL-7delta3/4、pET21b-delta4/5、pET21b-IL-7delta3/4/5. Theresultant recombinant expression vectors were transformed into E.coli BL21（DE3）. Afterinducing by IPTG, we using SDS-PAGE and western blot to analysis the protein.And theproteins were target proteins.And then we used Internet to analysis the protein of in theopen reading frame of splice variants of IL-7and got somen data about them.