牦牛转铁蛋白基因克隆与原核表达
发布时间：2018-09-05 15:41
【摘要】：以成年天祝白牦牛肝脏组织为试验材料,根据牦牛Tf基因CDS区序列设计引物,利用PCR和逆转录PCR技术克隆获得天祝白牦牛转铁蛋白基因编码区序列,并结合生物信息学方法对其遗传特性进行了分析；通过基因工程手段,构建原核重组表达载体,实现了牦牛转铁蛋白在E.coil BL21(DE3)中的异源表达；采用实验室3L发酵罐水平进行发酵小试,为深入研究转铁蛋白结构和功能,实现规模化工业生产提供实践基础和理论指导。研究结果如下：1.克隆获得了大小为2115bp的基因片段(GenBank收录号为KP720624),为Tf基因的完整编码区,编码704个氨基酸,其N端有一个19个氨基酸组成的信号肽,含两个高度保守的同源结构域,同源性为42%,Tf基因编码的蛋白分子量大小75.78KDa,理论等电点为6.63；2.序列分析显示,克隆获得的Tf基因序列存在4个碱基突变,其中：两个同义突变(57 A←→G,750 T←→c),两个错义突变(482 A←→T,511 T←→c),引起第161、171位氨基酸发生改变(L→G、L→F)。3.利用I)NAman软件进行氨基酸同源性分析,结果表明：天祝白牦牛Tf基因氨基酸序列与野牦牛、牛、水牛、藏羚羊、绵羊、野猪、人、鼠、鸡和鱼Tf氨基酸序列间的同源性分别为：100%、99%、96%、94%、93%、75%、69%、64%、51%、39%;4.在不改变氨基酸编码序列前提下,充分考虑并且适当的调整GC含量,对目的蛋白序列中阻碍蛋白翻译起始的二级结构和存在于目的蛋白序列中的原核宿主菌的稀有密码子进行优化,去除Tf自身的编码信号肽氨基酸序列。优化后的Tf基因序列GC含量为50%,优化了起始密码子附近mRNA的2个小的发夹结构和2个中等大小的发夹结构。通过全基因合成获得目的基因,构建重组子pGH-DT1219-Tf。5.构建Tf原核表达载体pET-30a-Tf。以E.coil BL21(DE3)为宿主菌,IPTG为诱导剂,筛选出最佳表达条件(18℃,IPTG终浓度为0.5mM,16h)诱导表达,经SDS-PAGE鉴定其分子量约为76 kDa。进行实验室3L发酵罐水平高密度发酵,经His-bind镍柱亲和层析和离子交换层析纯化获得浓度为0.80mg/mL,纯度大于70%的重组蛋白7.20mg。
[Abstract]:Using the liver tissue of adult Tianzhu white yak as experimental material, primers were designed according to the sequence of CDS region of yak Tf gene, and the coding region of transferrin gene of Tianzhu white yak was cloned by PCR and reverse transcription PCR. The genetic characteristics were analyzed by bioinformatics, and the recombinant expression vector was constructed by genetic engineering to realize the heterologous expression of yak transferrin in E.coil BL21 (DE3). In order to study the structure and function of transferrin and realize large-scale industrial production, the fermenting experiment was carried out at the level of 3L fermenter in laboratory, which provided the practical foundation and theoretical guidance for further studying the structure and function of transferrin. The results are as follows: 1. A gene fragment of 2115bp size (GenBank accession number is KP720624) was obtained, which is the complete coding region of Tf gene and encodes 704 amino acids. Its N-terminal has a signal peptide composed of 19 amino acids and contains two highly conserved homologous domains. The molecular weight of protein encoded by TF gene is 75.78KDa.The theoretical isoelectric point is 6.63kDa. Sequence analysis showed that there were four base mutations in the cloned Tf gene. Among them, two synonymous mutations (57A ~ (510T) and two missense mutations (~ (482A) (~ (482A) T ~ (511) T ~ + c),) resulted in the amino acid change (L ~ (161171) G ~ + L ~ (F) 路3 ~ (-1). The amino acid sequence of Tf gene in Tianzhu white yak was compared with that of wild yak, buffalo, Tibetan antelope, sheep, wild boar, human, mouse, chicken and fish. The results showed that the amino acid sequence of Tf gene in Tianzhu white yak and wild yak, buffalo, Tibetan antelope, sheep, wild boar, human, mouse, chicken and fish were respectively. Without changing the amino acid coding sequence, the content of GC was fully considered and adjusted appropriately. The secondary structure of the target protein which hinders the initiation of protein translation and the rare codon of the prokaryotic host bacteria in the target protein sequence were optimized to remove the amino acid sequence of the signal peptide of Tf itself. The GC content of the optimized Tf gene sequence was 50. The two small hairpin structures and two moderate hairpin structures of mRNA near the start codon were optimized. Construction of Recombinant pGH-DT1219-Tf.5. by Total Gene Synthesis Construction of Tf prokaryotic expression vector pET-30a-Tf. Using E.coil BL21 (DE3) as the inducer, the best expression conditions were selected. The final concentration of IPTG was 0.5 mm ~ (-1) 16h at 18 鈩，
本文编号：2224717
[Abstract]:Using the liver tissue of adult Tianzhu white yak as experimental material, primers were designed according to the sequence of CDS region of yak Tf gene, and the coding region of transferrin gene of Tianzhu white yak was cloned by PCR and reverse transcription PCR. The genetic characteristics were analyzed by bioinformatics, and the recombinant expression vector was constructed by genetic engineering to realize the heterologous expression of yak transferrin in E.coil BL21 (DE3). In order to study the structure and function of transferrin and realize large-scale industrial production, the fermenting experiment was carried out at the level of 3L fermenter in laboratory, which provided the practical foundation and theoretical guidance for further studying the structure and function of transferrin. The results are as follows: 1. A gene fragment of 2115bp size (GenBank accession number is KP720624) was obtained, which is the complete coding region of Tf gene and encodes 704 amino acids. Its N-terminal has a signal peptide composed of 19 amino acids and contains two highly conserved homologous domains. The molecular weight of protein encoded by TF gene is 75.78KDa.The theoretical isoelectric point is 6.63kDa. Sequence analysis showed that there were four base mutations in the cloned Tf gene. Among them, two synonymous mutations (57A ~ (510T) and two missense mutations (~ (482A) (~ (482A) T ~ (511) T ~ + c),) resulted in the amino acid change (L ~ (161171) G ~ + L ~ (F) 路3 ~ (-1). The amino acid sequence of Tf gene in Tianzhu white yak was compared with that of wild yak, buffalo, Tibetan antelope, sheep, wild boar, human, mouse, chicken and fish. The results showed that the amino acid sequence of Tf gene in Tianzhu white yak and wild yak, buffalo, Tibetan antelope, sheep, wild boar, human, mouse, chicken and fish were respectively. Without changing the amino acid coding sequence, the content of GC was fully considered and adjusted appropriately. The secondary structure of the target protein which hinders the initiation of protein translation and the rare codon of the prokaryotic host bacteria in the target protein sequence were optimized to remove the amino acid sequence of the signal peptide of Tf itself. The GC content of the optimized Tf gene sequence was 50. The two small hairpin structures and two moderate hairpin structures of mRNA near the start codon were optimized. Construction of Recombinant pGH-DT1219-Tf.5. by Total Gene Synthesis Construction of Tf prokaryotic expression vector pET-30a-Tf. Using E.coil BL21 (DE3) as the inducer, the best expression conditions were selected. The final concentration of IPTG was 0.5 mm ~ (-1) 16h at 18 鈩，
本文编号：2224717
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