Cloning, Expression Of Lamininα₅ LG1-3,LG2,LG3 CDNA And Generation Of Polyclonal Antibody Of LG1-3

Laminins are the major noncollagenous glycoproteins of all basal laminae (Bis). They are α/β/γ heterotrimers assembled from 10 known chains, and they subserve both structural and signaling roles. The 5 chain is present in virtually all BLs of early somite stage embryos and then becomes restricted to specific BLs as development proceeds, including those of the surface ectoderm and placental vasculature.In this experiment, total. RNAs were isolated from skeletal muscle, cardiac muscle, liver, and kidney of 7 days mice using Trizol reagent. Using a sensitive reverse transcription-polymerase chain reaction (RT-PCR) , it was found that the expressions of mRNAs were detected in skeletal muscle, cardiac muscle , kidney and liver tissues. The result was coincident with the published literatures.The cDNA fragments LG1-3, LG2, LG3 of LNas was amplified by RT-PCR, using total RNA of cardiac muscle as template and primers spanning the coding sequence of LNa 5 (Pl-3.1, Pl-3.2; P2.1, P2. 2; P3.1, P3.2). The resulted cDNA fragment with the expected size was cloned into vector pMD 18-T and subsequently subjected to restriction enzyme analysis and sequencing . The result showed that the cloned LG1-3, LG2, LG3 gene fragments are 1683bp, 555bp, 528bp comprising the complete coding sequence of LN and 100% homogenous to published data .Three PCRs were performed using the pairs of primers and the total mRNA of cardiac muscle. And three cDNAs fragment of 1683 bp, 555bp, 528bpwere obtained which include the complete coding sequence of LG1-3, LG2, LG3 of LN. Then the constructs of pMD18-T~LGl-3, pMD18-T-LG2, pMD18-T-LG3 were generated by inserting the three cDNA fragments into pMD18~T vector and selecting the sense clones by restriction enzyme analysis. Three constructs were digested with Xhol and Bam H I and ligated the pET28a(+) vector digested with the same enzymes using T4 DNA ligase. And three constructs of pET28a(+)-LG1-3, pET28a(+)-LG2 pET28a(+)HLG3 were generated by transforming the competent cell of BL21(DE3) of E. coli. The sense clones were obtained by restriction enzyme analysis. The plasmids of the constructs of pET28a(+)LGl-3,pET28a(+)-LG2 pET28a (+)-LG3 were sequenced and the three sequences were confirmed to be right. Incubate the genetic engineering bacteria in LB medium (KANr) and induce the expression of LN protein with IPTG at a final concentration of 1mM. Fortunately, all expressions of these LN fragments protein were observed on 12% SDS-PAGE, and the protein of LG3 was about 30% in all proteins of E. coli.The protein expressed by the plasmid of pET28a(+) LG1-3 in E. coli was purified and absolved in urea, then the solution was injected in the adult rabbits four times in two months. Then the blood was obtained from the rabbits and analyzed by ELISA, The result was very good.