The Construction Of CDNA Library Of Human Placenta Tissue And Cloning Of Human Growth Hormone

cDNA library construction and bolting are one of the important methods of gene cloning.Up to date,it is the basic tool of finding new genes and researching gene function.Target genes can be selected from cDNA library,and used to analyze the expression of the genes.The cloned fragments from classical cDNA library is short.However,full-length cDNA library can provide the completed information of mRNAs, so the full-length sequence can be cloned from it. In this study, extracting total RNA using TRIzol reagent from human placenta tissue, estimating the quality of cDNA library which constructed by long-distance LD-PCR, to clone human growth hormone (hGH) cDNA and it provide theory and technology basic for recombining human growth hormone. The results are as follows.1. Extracting totol RNA: put 100 mg tissue and 1ml TRIzol in cold grind-utersil, grind, centrifiguate, extract after adding 200 ul chloroform, transter the supernatant to a new eppendorf tube, then obtain pellet by adding the same volume cymene to the supernatant, dissolve this pellet by adding RNase-free H2O, extract after adding 1/10 volume NaAr and the same volume chloroform again and again until no protein like foam, obtain pellet by adding the same volume cymene, rinse the pellet by cold 70% ethanol twice, at last, dissolve the pellet with 30-50 ul DEPC H2O, conserve in -70℃.2.To construcd a cDNA library of tufted deer(placenta tissue ) testis,the total RNA was extracted using TRIzol reagent as the template , and F1,R2,F3,R4 as the two couples specific primer, we obtained the double strand cDNA through the effect of RT-PCR and polymerase. Then double strand cDNA and JG45 plasmid vector were cuted by the Sfi, and were linked by the T4 DNA joinase to complete the cDNA library. The qualities of the cDNA library were analyzed.3.The average capacities of the cDNA library was about 7.01×105 clones perμg ds-cDNA with recombinant rate of 96%.Among the 80 detected randomly selected cDNA clones, none repeated inserted cDNA fragment were founded .cDNA library construced with this method is suitable for functional genomic analyzing.4.Total RNA were isolated from human placenta tissue, then used RT-PCR to obtain the full-length hGH cDNA sequence.Compared the sequence which obtained from the constructed cDNA library using the PCR. They were confirmed be the same sequence, so cDNA library is available using this method.