Cloning And Expression Of Leptin, Ghrelin And IL-15 Gene And CDNA Library Construction Of Stomach And Spleen Of The Giant Panda

Giant panda(Ailuropoda melanoleuca),famous as the "living fossil",is an endangered species that endemic to China.For giant panda,due to its high specialization upon feeding habits,reproductive capability and cup-raising behavior,it has a typical low fertility and high infant mortality which make giant panda result in an endangered status. There are only about 1600 giant pandas surviving in the wild.Previous studies suggested that there is a seirious relationship beteewn the nutritional status and reproduction of the mammals.The leptin and Ghrelin participate in the control of reproduction,in conjunction with that of food intake and energy expenditure.Up to now,concerning the molecular mechanism underlying low fertility and high infant mortality is still poorly understood.Accordingly,cloning GH,Ghrelin,Leptin and IL-15 is important for understanding reproductive problems,puberty onset and field salvation of giant panda.It is harmful to population reconstruction for the small population,exhausted genetic resources and genetic degradation.So,construction gene library,such as cDNA library,is an effective methdod to conservation the genetic resources of the giant panda.This project is aimed at:(1) Cloning and expression of Ghrelin,Leptin and IL15 which play vital roles in growth,reproduction and deseases.(2) Construction cDNA library of stomach and spleen.The results obtained are as following:1.The complete coding sequences of Ghrelin,Leptin and Interleukin-15 were cloned from giant panda.The variants of the Leptin and Ghrelin mRNA were obtained.The coding sequences of Ghrelin and Ghrelin-des-Gln14 are 354bp and 351bp and encode an 117aa and an 116aa prepro-protein,the matural protein are 28aa and 27aa,respectively. The encoding sequence of Leptin and Leptin-3-deleted are 504bp and 501bp and encode an 167aa and an 166aa prepro-protein,the mature protein are 146aa and 145aa, respecitvely.The encoding sequence of IL-15 is 489bp and encodes an 162aa prepro-protein,the matural protein is 114aa.Analysis of the amino acid sequences indicated that Ghrelin,Leptin and Interleukin-15 have the most similarity to that of cat, bear and dog,respectively.Giant panda Ghrelin was 90.6%and 86.3%homologous to cat and dog,leptin was 99.4%,94.6%and 92.8%homologous to bear,dog and cat,IL-15 was 87.0%and 82.7%homologous to dog and cat.All the above suggested that the giant panda has the most closely relationship to cat,bear and dog.The tertiary structure analysis of Leptin and IL15 indicated that tertiary structure of giant panda IL15 protein was similary to Human IL15 protein,though giant panda Leptin had the same conservative doman sequences as the human Leptin,their tertiary structure is quite differenet.2.E.coli and Yeast expression system of giant pand genes were established.First, the mature peptide of giant panda growth hormone(AmGH) was successfully expressed and secreted in Pichia pastoris.The expression condition for AmGH was optimized.The secreted nascent AmGH were purified using ammonium sulfate fractionation.Then,as the working routine of expression AmGH in Pichia,the cDNA sequences ecoding the mature Leptin and IL-15 proteins were subcloned into pET28a and pPIC9K,and high expressed in E.coli BL21(DE3) and P.pastrois GS115.The protein purified by Affinity chromatography methord.Leptin gene contains 7 rare codons of 5 AGG,one AGA,and ATA,and IL15 contains 4 rare codons of 3 ATA,one TGT.The BL21(DE3)/pET28a-Leptin or BL21(DE3)/pET28a-IL15 can not expression the target gene effectively.The Leptin and IL15 were over-expression by Co-transformation the pSJS1240 and pET28a-Leptin or pET28a-IL15 into E.coli BL21(DE3).These studies would provide a facilitate studies on the biological activity of the three genes.3.The mRNA expression of Ghrelin in different issues such as intestine,liver, kidney,thymus,spleen and heart,was detected by RT-PCR.The observation is in line with previous data indicating that the stomach is the main expression and production site of ghrelin.Western blotting assy of the expression IL-15 was suggested that IL15 has a potential use of vaccine adjuvant for giant panda.The results will lay a foundation for a further study to treat hypoleptinemic and apply them in artificial breeding. 4.The full longth cDNA library of stomach and spleen were constructed successfully.The the titer of the unamplified libraries was 3.3×10~6 and 4.3×10~6,the percentage of recombinant clones was 85%and 82%.The titer of the amplified libraries was 6.9×10~9 and 6.4×10~9,respectively.The construction of these cDNA libraries provides a basis for further studies on the function genes of feeding habits and deseases.