【摘要】：以成年天祝白牦牛肝臟組織為試驗材料,根據(jù)牦牛Tf基因CDS區(qū)序列設(shè)計引物,利用PCR和逆轉(zhuǎn)錄PCR技術(shù)克隆獲得天祝白牦牛轉(zhuǎn)鐵蛋白基因編碼區(qū)序列,并結(jié)合生物信息學(xué)方法對其遺傳特性進(jìn)行了分析；通過基因工程手段,構(gòu)建原核重組表達(dá)載體,實現(xiàn)了牦牛轉(zhuǎn)鐵蛋白在E.coil BL21(DE3)中的異源表達(dá)；采用實驗室3L發(fā)酵罐水平進(jìn)行發(fā)酵小試,為深入研究轉(zhuǎn)鐵蛋白結(jié)構(gòu)和功能,實現(xiàn)規(guī)模化工業(yè)生產(chǎn)提供實踐基礎(chǔ)和理論指導(dǎo)。研究結(jié)果如下：1.克隆獲得了大小為2115bp的基因片段(GenBank收錄號為KP720624),為Tf基因的完整編碼區(qū),編碼704個氨基酸,其N端有一個19個氨基酸組成的信號肽,含兩個高度保守的同源結(jié)構(gòu)域,同源性為42%,Tf基因編碼的蛋白分子量大小75.78KDa,理論等電點為6.63；2.序列分析顯示,克隆獲得的Tf基因序列存在4個堿基突變,其中：兩個同義突變(57 A←→G,750 T←→c),兩個錯義突變(482 A←→T,511 T←→c),引起第161、171位氨基酸發(fā)生改變(L→G、L→F)。3.利用I)NAman軟件進(jìn)行氨基酸同源性分析,結(jié)果表明：天祝白牦牛Tf基因氨基酸序列與野牦牛、牛、水牛、藏羚羊、綿羊、野豬、人、鼠、雞和魚Tf氨基酸序列間的同源性分別為：100%、99%、96%、94%、93%、75%、69%、64%、51%、39%;4.在不改變氨基酸編碼序列前提下,充分考慮并且適當(dāng)?shù)恼{(diào)整GC含量,對目的蛋白序列中阻礙蛋白翻譯起始的二級結(jié)構(gòu)和存在于目的蛋白序列中的原核宿主菌的稀有密碼子進(jìn)行優(yōu)化,去除Tf自身的編碼信號肽氨基酸序列。優(yōu)化后的Tf基因序列GC含量為50%,優(yōu)化了起始密碼子附近mRNA的2個小的發(fā)夾結(jié)構(gòu)和2個中等大小的發(fā)夾結(jié)構(gòu)。通過全基因合成獲得目的基因,構(gòu)建重組子pGH-DT1219-Tf。5.構(gòu)建Tf原核表達(dá)載體pET-30a-Tf。以E.coil BL21(DE3)為宿主菌,IPTG為誘導(dǎo)劑,篩選出最佳表達(dá)條件(18℃,IPTG終濃度為0.5mM,16h)誘導(dǎo)表達(dá),經(jīng)SDS-PAGE鑒定其分子量約為76 kDa。進(jìn)行實驗室3L發(fā)酵罐水平高密度發(fā)酵,經(jīng)His-bind鎳柱親和層析和離子交換層析純化獲得濃度為0.80mg/mL,純度大于70%的重組蛋白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，

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