Stable Expression Of The Human Secreted Form Klotho Protein In CHO Cells

Senescece is a biology procedure with plentiful component elements, and it is the display of performance reduction and disorder. More and more domestic and overseas scientists devote themselves to the research about the mechanism, and they have researched in the aspects of the apparatus, the whole organism, the cell and molecular since many years ago. A great deal of theories about the senescence have been put to the people, such as free radicals theory, cross linkage theory, somatic mutation theory et al. However there is not an all-around explanation or a consistent agreement in any aging theories. The discussion of the connection between gene and senescence has becoming a focus along with the advancement of the molecular biology technology in recent years. A lot of genes about aging had been discovered in succession. In 1997 it was the discovery of a novel gene, termed klotho, that becoming the new hope of senescence research. Defects in the klotho (kl) gene cause mice to die prematurely with a skein of disorders commonly found in the elderly humans, such as arteriosclerosis, osteoporosis, skin atrophy and emphysema. And the kl gene mouse is the first laboratory animal model whose phenotypes closely resemble human aging by a single gene mutation. So far, the accurate biological functions of the KL protein were unknowen, and the recombinant expression of the native human secreted form KL (hsKL) protein was not repoted. The KL fusion protein had been used to research their functions all the while, and the native KL protein still had not been obtained up to the present. However the functions may be difference between these proteins. So we should prepare to recombinant express the native hsKL protein with CHO cells, which provide the essential conditions for the reseach of the human KL protein. 1. Clone and identification of the hskl gene To confirm the function and mechanism of the kl gene and KL protein, we research the human secreted form KL protein (549 amino acids) because of the expression of the putative secreted form predominating over that of the membrane form in the human being. In our studies, the clone vector pBS-hskl was constructed by gene jointing from the human genome DNA. The human secreted form kl (hskl) gene exon 1 was obtained via nest PCR. The PCR products with the predicted size were further isolated and cloned into the pBluescript II vector. So the vector pBS-hsklex1 was constructed and the restriction site NheI was introduced into the vector successfully. The hskl exon 2 gene was subcloned into the above vector pBS-hsklex1. We obtained the vector pBS-hsklexon(1+2) that contained a new restriction site HindIII. The clone vector pBS-hskl was finally constructed by the same method. The hskl exon 3 gene was obtained via PCR, and it was subcloned into the above vector pBS-hsklexon(1+2). The aforementioned plasmids were all authenticated by sequencing. We modified the individual bases which won't change the amino acid for introducing the restriction site to joint the three exons. The restriction site NheI was introduced with 816c→g(Leu→Leu),819g→a(Leu→Leu)in the joint between exon1 and exon2. The restriction site HindIII was similarly introduced with 1341g→(tLeu→Leu)in the joint between exon2 and exon3. The sequencing showed that results were identical with the predicted ones. In our studies, the recombinant human secreted form klotho (rhskl) gene contained one SNP which displayed in the hskl sequence of GeneBank. The SNP is 281t→c(Asp→Asp), and the alteration of the base do not change the amino acid. 2. Construction and preparation by screening of the CHO strain with stable expression of the human secreted form Klotho protein The expression vector pcDNA3.1/Zeo(+)-hskl was constructed from the clone vector pBS-hskl by PCR. The plasmids were purified using Wizard Plus MinipripsDNA Purification System after sequencing. CHO cells were transfected stably by the expression vector pcDNA3.1/Zeo(+)-hskl via the Polybrene transfection reagent. The positive clone cells were cultured scale-up with DMEM medium containing 300μg/ml Zeocin. The cell lines that stably expressing the hsKL protein were obtained, namely CHO-hsKL. The DMEM medium without serum and antibiotics was used when the cells was confluent more than 90% in the clutrued dishes. The culture supernatant of CHO-hsKL was gathered per 24 hours, and was centrifuged to eliminate the cells debris. Then the gathered supernatant with 50μg/ml p-APMSF was kept at -80℃. 3. Identification of the stable transfected cell strain CHO-hsKL 1) PCR in the CHO-hsKL cell strain The genome DNA prepared from CHO-hsKL, producers of hskl gene, was amplified by LA-Taq DNA polymerase using specific primers for hskl gene. The resulting DNA was only showed in the transfected cells, and there is not the predicted DNA in the control groups (CHO cells without the transfected klotho gene and CHO cells with the transfected blank plasmid). This indicated that hskl gene was stably transfected into CHO genome DNA. 2) SDS-PAGE of the hsKL protein The supernatant concentrated via lyophilization was analyzed by SDS-PAGE (argentation). We seen that the 66kDa protein bands only existed in the supernatant of stably transfected CHO-hsKL. 3) Western blot of the hsKL protein The supernatant concentrated via lyophilization was analyzed by western blot. The resulting protein bands was only showed in the transfected cells, and there is not the predicted protein bands in the control groups, so we believed the human secreted form KL protein were obtained successfully. But there were different expression quantities among the clone cells. The reasons may be that: ①The extrinsic gene integrated into the different sites of the cell's chromosome, and the transcript efficiency was different. ②The copy numbers of the plasmids were different among the transformed cells.