Cloning Of Human Dipeptidyl Peptidase Ⅳ Gene Segments And Identification

The complete cDNA and derived amino acid sequence for human CD-26 was first published in 1992.The CD26 gene encodes a type II trans-membrane protein of 766 amino acids, which is anchored to the lipid bila-yer by a single hydrophobic segment locatedat the N-terminus,and has ashort cytoplasmic tail of six amino acids.A flexible stalk links the memb-raneanchor to a large glycosylated region,a cysteine-rich region and a C-terminal catalytic domain.Alignment of the amino acid sequences reveals a high degree of conservation between differentspecies,with the C-termina-l segment showing the highest level of identity.The active site Ser-630 of human CD26 is surrounded by Gly-Trp-Ser630-Tyr-Gly-Gly-Tyr-Val,wh-ich corresponds to the motif Gly-X-Ser-X-Gly proposed for serine-type pe-ptidases.A more extended consensus motif around the catalytic serine is shared by all members of the prolyl oligopeptidase family,to which CD2-6 belongs.The linear order of the catalytic triad in this family of peptida-ses is nucleophileacid-base (Ser-Asp-His),an arrangement not common fo-r"classic"serine-type peptidases (trypsin or chymotrypsin-like enzymes) but characteristic of theα/βhydrolase fold.This enzyme is a dipeptidase t-hat preferentially cleaves peptides from the N-terminus with the sequenceGly-Pro-Xaa or Ala-Pro-Xaa in the penultimate position.DPPIV is expressed relatively abundantly in several tissues including small intestine, lung and kidney.It was shown to contain 26 exons,that the 5'-flanking region of the DPP IV gene is GC-rich.The first 500 base-s upstream of the initiation codon contains 71% G+C residues.Moreover,it contains high content of the rare CpG dinucleotide,the DPP IV promot -er resides within an unmethylated CpG island.Thisis characteristic of thepromoters found in housekeeping genes,the DPP IV 5'-flanking sequence has other features of a housekeeping gene promoter,it contains several Spl-binding sites and lacks a consensus TATA box,these characteristics ma-y play a role in the expression of the DPP IV transcript in multiple celltypes.Although the DPP IV promoter lacks a consensus TATA box,It c-ontains two AT-rich sequences that are deemed to be TATA-like seque-nces,the proximal TATA-like sequence (5'-TTTAACTC) may well functio-nto bind transcription factorIID (TATA-binding protein).There are many physiological function for CD26.For examble,it's e-xopeptidase activity,collagen and fibronectin binds to CD26,CD26 was i-dentified as the binding protein for adenosine deaminase (ADA),CD26 canactivate T cell and produce cytokine,It is now well recognized that HIV-1-infected individuals have significantly lower percentages of CD26bright cel-ls than non-infected controls.So we would construct prokaryotic expressi-on system to analysis the enzymatic activity of DPP4 and antigenicity.T-his job do the groundwork to profuondly study on the physiological funct-ions for CD26.First,we extracted the total RNA from the normal intestine of theint-estinal obstruction patient using the Trizol reagent and cloned the humanCD26 gene segments from E608 to H748 by RT-PCR,finally obtain a 44-3bp fragments.The purified product of PCR was inserted into pGEM?-Tvector,recombinants were indentified by blue/white screening on the LB plate,recombinant plasmid DNA were extracted from the white colonies a-nd sequenced.the result of sequencing was blastedto the Genbank databa -se,the result of feedback is right.We cloned the objective gene fragme-nts again using the plasmid DNA as the templates.Both the pET-28a ve-ctor and the product of PCR were digested with EcorⅠand HindⅢ,puri-fied the above DNA with the gel purification kit,inserted the PCR frag-ment into the pET-28a by theratios of 3:1 with T4 DNA ligase,screened-the positive colonies with kanamycin,recombinant plasmid DNA were ext-racted from the positive colonies and sequenced,the result of sequencing was blasted to the Genbank database,the result of feedback is right.We successfully cloned human CD26 gene fragments using RT-PCR,and the objective fragments were inserted into pET-28a.the recombinant plasmid is called pET-28a-hCD26,the result of sequencing showsthat the ORF is correct,we would have expressed the aim protein by IPTG induc-tion,analyzed the enzymatic activity and antigenicity by the following w-ork.