大熊猫Leptin、Ghrelin和IL-15基因的克隆表达及胃和脾脏cDNA文库的构建
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摘要
大熊猫(Ailuropoda melanoleuca)是我国特有的国家Ⅰ一级重点保护野生动物,被誉为动物界的“活化石”。由于大熊猫自身的食性、繁殖能力和育幼行为的高度特化等原因,造成了大熊猫目前濒危状态,野生大熊猫现在仅存1600只左右。哺乳动物的繁殖和生长与营养状况密切相关,Leptin和Ghrelin就是将摄食、能量代谢和生殖生长联系起来的关键激素。由于对大熊猫生殖力低和初生幼仔发育不良的分子机制的研究还很薄弱,对大熊猫生长发育、疾病、繁殖相关基因(如GH、Leptin、Ghrelin和IL-15等基因)的克隆,对了解大熊猫生长发育规律,青春期发动,繁殖规律以及野外大熊猫的人工救助和保护具有重要的意义。大熊猫种群的规模小,遗传资源的枯竭和遗传退化都不利于种群的复壮与重建,尽快建立大熊猫的基因文库(包括cDNA文库与基因组文库等)不失为一种有效保存现有遗传资源的方法。本研究的目的就是克隆大熊猫Ghrelin、Leptin和IL-15等功能基因,并在原核和真核表达系统中实现表达;构建胃和脾脏等全长cDNA文库,为研究这些基因在大熊猫发育、繁殖中的作用及大熊猫基因资源的保存提供支持。
本研究获得以下主要结果:
1.克隆了大熊猫Ghrelin、Leptin和Interleukin-15的全长编码区,并鉴定得到Ghrelin和Leptin的两种不同mRNA剪接体。Ghrelin和Ghrelin-des-Gln14基因编码区序列为354bp和351bp,分别编码117和116氨基酸的前体蛋白,成熟蛋白分别为28和27个氨基酸;Leptin和Leptin-3-deleted编码区序列为504bp和501bp,分别编码167和166氨基酸的前体蛋白,成熟蛋白为146和145氨基酸;IL-15基因编码区序列为489bp,编码一个162氨基酸的前体蛋白,成熟蛋白为114氨基酸。氨基酸序列分析表明,大熊猫Ghrelin,与猫和狗的氨基酸序列相似性最高,分别高达90.6%和86.3%;Leptin与熊、狗和猫的相似性高达99.4%、94.6%和92.8%;IL-15与狗、猫的相似性高达87.0%和82.7%。以上表明大熊猫三个基因在进化上可能与狗、熊和猫亲缘关系最近。Leptin和IL15蛋白质结构模拟结果表明,大熊猫IL15的三级结构跟人的高度相似;尽管大熊猫Leptin蛋白保守域跟人的完全相同,但三级结构与人的有较大差异。
2.建立了大熊猫基因在大肠杆菌原核表达和酵母真核表达体系,首先以大熊猫GH基因进行了酵母真核分泌表达体系的建立,并系统的优化了GH分泌表达的条件,探索了用硫酸铵分级沉淀纯化表达产物的方法;然后采用优化的表达系统和纯化方法,实现了大熊猫Leptin和Interleukin-15在酵母表达系统中高效表达,用亲和柱对表达蛋白的纯化。Leptin基因含有5个AGG,1个AGA,1个ATA;IL-15基因含有3个ATA,一个TGT。这些密码子都是大肠杆菌稀有密码子。在单独使用BL21(DE3)-pET28a-gene表达系统时,菌株会生长会受到抑制,很难获得表达。本研究将质粒pSJS1240和pET28a-Leptin或pET28a-IL15共转化到E.coli BL21(DE3)菌株,kan和SP抗生素筛选,得到过表达菌株BL21(DE3)(pSJS1240/pET28a-Leptin)和BL21(DE3)(pSJS1240/pET28a-IL15),使这两个含大量稀有密码子的基因在大肠杆菌中获得成功表达;为这三个大熊猫基因的体外表达创建了技术平台,为深入研究基因功能奠定了基础。
3.用RT-PCR方法检测了Ghrelin在小肠、肝脏、肾脏、胸腺、脾脏和心脏组织的mRNA表达,表明大熊猫Ghrelin的最主要表达组织是胃,在小肠、肝脏和肾脏中只有较弱表达,而在胸腺、脾脏和心脏组织中没有检测到该基因的表达。另外,采用Western方法检测了表达的大熊猫IL-15蛋白的免疫原性,结果表明IL-15具有作为大熊猫疫苗佐剂的潜力;Leptin可以作为大熊猫Leptin缺乏引起的各种疾病的防治,应用于人工繁育大熊猫。
4.构建了胃和脾脏的全长cDNA文库。初级文库的滴度分别为3.3×10~6和4.3×10~6;重组率分别达到85%和82%;扩增文库的滴度分别为6.9×10~9和6.4×10~9。全长文库的构建为大熊猫食性和疾病相关功能基因的挖掘和研究提供了基础。
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.
本研究获得以下主要结果:
1.克隆了大熊猫Ghrelin、Leptin和Interleukin-15的全长编码区,并鉴定得到Ghrelin和Leptin的两种不同mRNA剪接体。Ghrelin和Ghrelin-des-Gln14基因编码区序列为354bp和351bp,分别编码117和116氨基酸的前体蛋白,成熟蛋白分别为28和27个氨基酸;Leptin和Leptin-3-deleted编码区序列为504bp和501bp,分别编码167和166氨基酸的前体蛋白,成熟蛋白为146和145氨基酸;IL-15基因编码区序列为489bp,编码一个162氨基酸的前体蛋白,成熟蛋白为114氨基酸。氨基酸序列分析表明,大熊猫Ghrelin,与猫和狗的氨基酸序列相似性最高,分别高达90.6%和86.3%;Leptin与熊、狗和猫的相似性高达99.4%、94.6%和92.8%;IL-15与狗、猫的相似性高达87.0%和82.7%。以上表明大熊猫三个基因在进化上可能与狗、熊和猫亲缘关系最近。Leptin和IL15蛋白质结构模拟结果表明,大熊猫IL15的三级结构跟人的高度相似;尽管大熊猫Leptin蛋白保守域跟人的完全相同,但三级结构与人的有较大差异。
2.建立了大熊猫基因在大肠杆菌原核表达和酵母真核表达体系,首先以大熊猫GH基因进行了酵母真核分泌表达体系的建立,并系统的优化了GH分泌表达的条件,探索了用硫酸铵分级沉淀纯化表达产物的方法;然后采用优化的表达系统和纯化方法,实现了大熊猫Leptin和Interleukin-15在酵母表达系统中高效表达,用亲和柱对表达蛋白的纯化。Leptin基因含有5个AGG,1个AGA,1个ATA;IL-15基因含有3个ATA,一个TGT。这些密码子都是大肠杆菌稀有密码子。在单独使用BL21(DE3)-pET28a-gene表达系统时,菌株会生长会受到抑制,很难获得表达。本研究将质粒pSJS1240和pET28a-Leptin或pET28a-IL15共转化到E.coli BL21(DE3)菌株,kan和SP抗生素筛选,得到过表达菌株BL21(DE3)(pSJS1240/pET28a-Leptin)和BL21(DE3)(pSJS1240/pET28a-IL15),使这两个含大量稀有密码子的基因在大肠杆菌中获得成功表达;为这三个大熊猫基因的体外表达创建了技术平台,为深入研究基因功能奠定了基础。
3.用RT-PCR方法检测了Ghrelin在小肠、肝脏、肾脏、胸腺、脾脏和心脏组织的mRNA表达,表明大熊猫Ghrelin的最主要表达组织是胃,在小肠、肝脏和肾脏中只有较弱表达,而在胸腺、脾脏和心脏组织中没有检测到该基因的表达。另外,采用Western方法检测了表达的大熊猫IL-15蛋白的免疫原性,结果表明IL-15具有作为大熊猫疫苗佐剂的潜力;Leptin可以作为大熊猫Leptin缺乏引起的各种疾病的防治,应用于人工繁育大熊猫。
4.构建了胃和脾脏的全长cDNA文库。初级文库的滴度分别为3.3×10~6和4.3×10~6;重组率分别达到85%和82%;扩增文库的滴度分别为6.9×10~9和6.4×10~9。全长文库的构建为大熊猫食性和疾病相关功能基因的挖掘和研究提供了基础。
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.
引文
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