大熊猫γ-干扰素基因克隆、原核表达及真核表达载体构建
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摘要
γ-干扰素(IFN-γ)主要是由活化的Th_1细胞、CD_8~+T细胞和NK细胞产生，除具有抗病毒和抗肿瘤活性外，免疫调节作用是其主要的生物学功能，如：激活巨噬细胞、上调MHCI、Ⅱ类分子表达水平和促进抗原提呈，诱导Th细胞的分化等，在机体针对肿瘤及多种胞内病原体感染等疾病的细胞免疫中发挥重要的作用，是体现机体免疫功能的一项重要指标。
大熊猫(Ailuropoda melanoleuca)是我国特有的珍稀野生动物，被誉为中国的“国宝”。据国家林业总局最新公布的调查数字，野生大熊猫现存数量在1590只左右，人工圈养的大熊猫也仅有189只，它们仍处于绝灭的边缘。长期人工圈养的大熊猫(特别是幼年大熊猫和老龄化大熊猫)大多免疫力低下，抗病毒、细菌和寄生虫感染的能力相对较弱，对其疾病的防治目前仍以药物控制为主，并无专用疫苗，加上频繁用药导致耐药性菌(虫)株出现，以及药物自身存在的毒副作用，这些都给大熊猫的疾病防治及繁殖工作带来了很大的难度。因而为进一步搞好大熊猫的饲养管理及疾病控制，就需要研究新的防治技术，通过细胞因子的作用来提高大熊猫的自身免疫力将可能是一个相对有效的拯救措施之一。
1．本试验应用反转录-聚合酶链反应(RT-PCR)技术，从ConA诱导培养的大熊猫外周血淋巴细胞总RNA中扩增得到大熊猫γ-干扰素基因，并将其克隆到PGEMT载体中。经菌落PCR鉴定、序列测定及序列分析，克隆得到的IFN-γ基因开放阅读框由498个核苷酸组成，编码一个由166个氨基酸组成的多肽，包括编码19个氨基酸的信号肽和147个氨基酸的成熟肽。与Genbank中其他哺乳动物的IFN-γ序列比较，它与犬、猫、人、猪、牛、羊、马、兔、鼠等的核苷酸同源性在57.7％(鼠)～93.2％(犬)之间；IFN-γ编码氨基酸同源性在46.8％(鼠)～92.8％(犬)之间；进化树构建结果表明大熊猫与犬的亲缘关系最近。
2．本试验构建了IFN-γ基因全编码序列的原核表达质粒pET32aIFN-γ，并进行了原核表达研究。将克隆在PGEMT载体中的IFN-γ基因全编码序列插入原核表达载体pET32a，得到重组质粒pET32aIFN-γ，转化大肠杆菌BL21(表达菌株)，IPTG诱导表达后，裂解菌体作SDS-PAGE分析，得到融合表达蛋白特异条带，该表达条带大小为34.6KDa(IFN-γ成熟蛋白为17.4 KDa，菌蛋白为17.2 KDa)。
3．将克隆在PGEMT载体中的IFN-γ基因全编码序列经EcoRI和XhoI酶进行双酶切，酶切产物经琼脂糖凝胶电泳，采用胶回收试剂盒回收纯化酶切产物，然后与同样酶切的pcDNA3.1(+)质粒定向连接并转化到大肠杆菌中，构建真核表达质粒pcDNA3.1(+)IFN-γ，并用酶切分析和序列测定进行了鉴定，结果酶切分析和序列测定都证实真核表达质粒成功构建。
本研究成功克隆了大熊猫IFN-γ基因，构建了原核和真核表达质粒，并进行了原核蛋白的表达。本研究为了解各物种间干扰素序列的进化关系提供理论依据，同时也为进一步研究IFN-γ重组蛋白的免疫学特性和生物学活性及其应用奠定了基础。
Abstract: Interferon-gamma(IFN-γ), produced mainly by Th_1 cells, CD_8~+T cells and natural killer cells, plays a critical role in anti-virus, anti-tumor, and immune modulation. Its immune modulation includes activating macrophages, up-regulating the expression of MHC classⅠand classⅡmolecules, prompting the presentation of antigens, and inducing Th_1 cell growth and differentiation. The level of IFN-γis an important index to the cellular immunity.
Giant panda is a characteristic and precious wildlife in our country,it is named "national treasure" in China.The investigation from Nation Forestry Head Office indicates that the number of wild Giant panda is about 1590, captive Giant panda is only 189, they are still faced to extinction. The long-term captive giant pandas ,especially baby or aging Giant panda with hypoimmunity, are easily infected by virus, germ and parasite.The way of preventing or curing disease is mainly by drug,no special vaccine,but the drug would benoxious for giant panda with adverse reaction and the pathogen would become drug-resistant. All of these are bad for disease prevention and breeding of Giant panda,so we need new technologies to breed giant panda and control diseases, the way of improving autoimmunity by cytokines may be one effective rescueing measure relatively.
1. By reverse transcription polymerrase chain reaction(RT-PCR), Giant panda IFN-γgene was cloned from total RNA which was extracted from ConA-stimulated-cultured peripheral blood lymphocytes of giant panda, the amplified products of PCR were cloned into PGEMT vector.Alter identification and sequencing,the sequence analysis revealed that the open reading frame of giant panda IFN-γgene contained 498 nucleotides which encoding 166 amino acids,the signal peptide contained 19 amino acids and the mature IFN-γgene was composed of 147 amino acids. Compared with other mammals in Genbank, including dog, cat, human, pig, cow, horse, goat, rabbit, rat and so on, the homology of nucleotide sequence was between 57.7 % (rat) and 93.2%(dog), the homology of amino acid sequence was between 46.8%(rat) and 92.8 %(dog). Phylogenetic analysis based on the nucleotide sequence from the IFN-γgene showed that Giant panda has the most closed relationgship genetic distance with dog.
2. In this study, pET32alFN-γ, the prokaryotic expression plasmid with the complete sequence of IFN-γgene ,was constructed and expressed the corresponding protein. The complete sequence of IFN-γgene from PGEMT was subcloned into prokaryotic expression vetor pET32a,then a recombinant plasmid pET32aIFN-γwas obtained. The plasmid pET32aIFN-γwas transformed into E.coli.BL21 (expression E.coli ).The target protein was detected by SDS-PAGE analysis after induction by IPTG. Then the special band of fusion expressed protein was roughly obtained,and it was approximately 34.6KDa ( target protein and E.coli protein were 17.4 KDa and 17.2 KDa respectively ).
3. The complete sequence of IFN-γgene from PGEMT was double cleaved by EcoRI and XhoI, the cleaved product was reclaimed by DNA UNIQ-10 spin column purification kit after gel electrophoresis,then ligated with pcDNA3.1(+) plasmid double cleaved with the same enzyme and transformed into E.coli, pcDNA3.1(+)IFN-γwas identitied by cleaving analysis and sequencing,the result indicated that the eukaryotic expression plasmid was constructed successfully.
IFN-γgene was cloned and expressed in prokaryocyte successfully, and the eukaryotic expression plasmid was constructed. It would provide some evidence for the evolutional relationship of different species, Meanwhile, it provided a basis for further research on immunological and molecular biologic activity and its amplification of recombinant IFN-γprotein.
大熊猫(Ailuropoda melanoleuca)是我国特有的珍稀野生动物，被誉为中国的“国宝”。据国家林业总局最新公布的调查数字，野生大熊猫现存数量在1590只左右，人工圈养的大熊猫也仅有189只，它们仍处于绝灭的边缘。长期人工圈养的大熊猫(特别是幼年大熊猫和老龄化大熊猫)大多免疫力低下，抗病毒、细菌和寄生虫感染的能力相对较弱，对其疾病的防治目前仍以药物控制为主，并无专用疫苗，加上频繁用药导致耐药性菌(虫)株出现，以及药物自身存在的毒副作用，这些都给大熊猫的疾病防治及繁殖工作带来了很大的难度。因而为进一步搞好大熊猫的饲养管理及疾病控制，就需要研究新的防治技术，通过细胞因子的作用来提高大熊猫的自身免疫力将可能是一个相对有效的拯救措施之一。
1．本试验应用反转录-聚合酶链反应(RT-PCR)技术，从ConA诱导培养的大熊猫外周血淋巴细胞总RNA中扩增得到大熊猫γ-干扰素基因，并将其克隆到PGEMT载体中。经菌落PCR鉴定、序列测定及序列分析，克隆得到的IFN-γ基因开放阅读框由498个核苷酸组成，编码一个由166个氨基酸组成的多肽，包括编码19个氨基酸的信号肽和147个氨基酸的成熟肽。与Genbank中其他哺乳动物的IFN-γ序列比较，它与犬、猫、人、猪、牛、羊、马、兔、鼠等的核苷酸同源性在57.7％(鼠)～93.2％(犬)之间；IFN-γ编码氨基酸同源性在46.8％(鼠)～92.8％(犬)之间；进化树构建结果表明大熊猫与犬的亲缘关系最近。
2．本试验构建了IFN-γ基因全编码序列的原核表达质粒pET32aIFN-γ，并进行了原核表达研究。将克隆在PGEMT载体中的IFN-γ基因全编码序列插入原核表达载体pET32a，得到重组质粒pET32aIFN-γ，转化大肠杆菌BL21(表达菌株)，IPTG诱导表达后，裂解菌体作SDS-PAGE分析，得到融合表达蛋白特异条带，该表达条带大小为34.6KDa(IFN-γ成熟蛋白为17.4 KDa，菌蛋白为17.2 KDa)。
3．将克隆在PGEMT载体中的IFN-γ基因全编码序列经EcoRI和XhoI酶进行双酶切，酶切产物经琼脂糖凝胶电泳，采用胶回收试剂盒回收纯化酶切产物，然后与同样酶切的pcDNA3.1(+)质粒定向连接并转化到大肠杆菌中，构建真核表达质粒pcDNA3.1(+)IFN-γ，并用酶切分析和序列测定进行了鉴定，结果酶切分析和序列测定都证实真核表达质粒成功构建。
本研究成功克隆了大熊猫IFN-γ基因，构建了原核和真核表达质粒，并进行了原核蛋白的表达。本研究为了解各物种间干扰素序列的进化关系提供理论依据，同时也为进一步研究IFN-γ重组蛋白的免疫学特性和生物学活性及其应用奠定了基础。
Abstract: Interferon-gamma(IFN-γ), produced mainly by Th_1 cells, CD_8~+T cells and natural killer cells, plays a critical role in anti-virus, anti-tumor, and immune modulation. Its immune modulation includes activating macrophages, up-regulating the expression of MHC classⅠand classⅡmolecules, prompting the presentation of antigens, and inducing Th_1 cell growth and differentiation. The level of IFN-γis an important index to the cellular immunity.
Giant panda is a characteristic and precious wildlife in our country,it is named "national treasure" in China.The investigation from Nation Forestry Head Office indicates that the number of wild Giant panda is about 1590, captive Giant panda is only 189, they are still faced to extinction. The long-term captive giant pandas ,especially baby or aging Giant panda with hypoimmunity, are easily infected by virus, germ and parasite.The way of preventing or curing disease is mainly by drug,no special vaccine,but the drug would benoxious for giant panda with adverse reaction and the pathogen would become drug-resistant. All of these are bad for disease prevention and breeding of Giant panda,so we need new technologies to breed giant panda and control diseases, the way of improving autoimmunity by cytokines may be one effective rescueing measure relatively.
1. By reverse transcription polymerrase chain reaction(RT-PCR), Giant panda IFN-γgene was cloned from total RNA which was extracted from ConA-stimulated-cultured peripheral blood lymphocytes of giant panda, the amplified products of PCR were cloned into PGEMT vector.Alter identification and sequencing,the sequence analysis revealed that the open reading frame of giant panda IFN-γgene contained 498 nucleotides which encoding 166 amino acids,the signal peptide contained 19 amino acids and the mature IFN-γgene was composed of 147 amino acids. Compared with other mammals in Genbank, including dog, cat, human, pig, cow, horse, goat, rabbit, rat and so on, the homology of nucleotide sequence was between 57.7 % (rat) and 93.2%(dog), the homology of amino acid sequence was between 46.8%(rat) and 92.8 %(dog). Phylogenetic analysis based on the nucleotide sequence from the IFN-γgene showed that Giant panda has the most closed relationgship genetic distance with dog.
2. In this study, pET32alFN-γ, the prokaryotic expression plasmid with the complete sequence of IFN-γgene ,was constructed and expressed the corresponding protein. The complete sequence of IFN-γgene from PGEMT was subcloned into prokaryotic expression vetor pET32a,then a recombinant plasmid pET32aIFN-γwas obtained. The plasmid pET32aIFN-γwas transformed into E.coli.BL21 (expression E.coli ).The target protein was detected by SDS-PAGE analysis after induction by IPTG. Then the special band of fusion expressed protein was roughly obtained,and it was approximately 34.6KDa ( target protein and E.coli protein were 17.4 KDa and 17.2 KDa respectively ).
3. The complete sequence of IFN-γgene from PGEMT was double cleaved by EcoRI and XhoI, the cleaved product was reclaimed by DNA UNIQ-10 spin column purification kit after gel electrophoresis,then ligated with pcDNA3.1(+) plasmid double cleaved with the same enzyme and transformed into E.coli, pcDNA3.1(+)IFN-γwas identitied by cleaving analysis and sequencing,the result indicated that the eukaryotic expression plasmid was constructed successfully.
IFN-γgene was cloned and expressed in prokaryocyte successfully, and the eukaryotic expression plasmid was constructed. It would provide some evidence for the evolutional relationship of different species, Meanwhile, it provided a basis for further research on immunological and molecular biologic activity and its amplification of recombinant IFN-γprotein.
引文
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