【摘要】：營養(yǎng)學(xué)研究表明，蛋白質(zhì)在人體內(nèi)主要以小肽的形式通過寡肽轉(zhuǎn)運(yùn)載體的轉(zhuǎn)運(yùn)被吸收。寡肽轉(zhuǎn)運(yùn)載體POT家族包含PepT1, PepT2, PHT1和PHT2四個(gè)成員,負(fù)責(zé)大多數(shù)二肽、三肽及其類似藥物的轉(zhuǎn)運(yùn)。其中，PepT2在很多器官中都有表達(dá)，包括腎臟、大腦、眼睛、乳腺等等。人類寡肽轉(zhuǎn)運(yùn)蛋白hPepT2包含729個(gè)氨基酸殘基、12個(gè)跨膜區(qū)和9跨膜區(qū)與10跨膜區(qū)之間的一個(gè)胞外大環(huán)，是一種高親和性、低容量的寡肽轉(zhuǎn)運(yùn)載體。氨基酸突變法和蛋白嵌合法研究表明，，人類寡肽轉(zhuǎn)運(yùn)蛋白hPepT2的1-9跨膜區(qū)決定其表現(xiàn)特征，7-9跨膜區(qū)為其底物結(jié)合區(qū)域，最后三個(gè)跨膜區(qū)仍為未知功能區(qū)域。 本文對(duì)人類寡肽轉(zhuǎn)運(yùn)蛋白10-11跨膜區(qū)片段hPepT2(579-664)進(jìn)行了原核表達(dá)。通過設(shè)計(jì)引物對(duì)hPepT2的cDNA進(jìn)行PCR克隆獲得目的基因，進(jìn)而通過限制性內(nèi)切酶進(jìn)行雙酶切和T4連接酶連接，構(gòu)建了pET30a(+)-hPepT2(579-664)重組質(zhì)粒。重組質(zhì)粒經(jīng)過測序無氨基酸突變，然后轉(zhuǎn)入E.coli BL21(DE3)pLysS中進(jìn)行表達(dá)。對(duì)含表達(dá)重組體系的菌液在37℃下，使用工作濃度0.3mM的IPTG進(jìn)行誘導(dǎo)表達(dá)，4h后獲得良好的表達(dá)效果。菌體沉淀再經(jīng)洗滌、超聲破菌、葡聚糖凝膠G-75柱純化、透析、冷凍干燥得到目標(biāo)蛋白。利用紫外可見光譜進(jìn)一步確定了蛋白的水溶性，利用紫外光譜和熒光光譜研究了其在不同pH緩沖液中的穩(wěn)定性，發(fā)現(xiàn)蛋白在pH7.5時(shí)較穩(wěn)定。 利用固相合成法合成了具有生物活性的十個(gè)含有酪氨酸的二肽：酪胺酰甘氨酸、酪胺酰亮氨酸、酪胺酰苯丙氨酸、酪胺酰賴氨酸、酪胺酰精氨酸、酪胺酰酪氨酸、絲氨酰酪氨酸、脯氨酰酪氨酸、纈氨酰酪氨酸、β-丙氨酰酪氨酸，并對(duì)合成產(chǎn)物進(jìn)行了高效液相分析、純化、ESI-MS表征。利用熒光光譜法研究了4種二肽與人類寡肽轉(zhuǎn)運(yùn)蛋白hPepT2(579-664)的相互作用，考察了肽濃度、作用時(shí)間對(duì)相互作用的影響。結(jié)果表明，二肽與濃度為2.52mg/ml蛋白相互作用，其最適濃度為3.33×10~(-3)-1.0×10~(-2)mg/ml，此時(shí)蛋白質(zhì)的熒光強(qiáng)度最大，同時(shí)二肽可以促使蛋白結(jié)構(gòu)發(fā)生改變，增加蛋白的熒光發(fā)射強(qiáng)度，隨著反應(yīng)時(shí)間的延長逐漸增加，最后趨于平衡。證明人類寡肽轉(zhuǎn)運(yùn)蛋白hPepT2(579-664)可以結(jié)合二肽或與二肽相互作用，二者之間存在相互作用。本研究為研究人類寡肽轉(zhuǎn)運(yùn)蛋白PepT2的功能和底物結(jié)構(gòu)以及新藥的設(shè)計(jì)提供了基礎(chǔ)數(shù)據(jù)。
[Abstract]:Nutritional studies show that proteins are mainly absorbed in the form of small peptides by the transport of oligoseptide transporters. The POT family of oligoseptide transporters consists of four members, PepT1, PepT2, PHT1 and PHT2, which are responsible for the transport of most dipeptides, tripeptides and their analogues. PepT2 is expressed in many organs, including the kidneys, brain, eyes, mammary glands and so on. Human oligoseptide transporter (hPepT2) contains 729 amino acid residues, 12 transmembrane regions and a large extracellular ring between 9 and 10 transmembrane regions, which is a high affinity and low capacity oligopeptide transport vector. The results of amino acid mutation and protein embedding showed that the 1-9 transmembrane region of human oligopeptide transporter (hPepT2) determined its characteristic transmembrane domain 7-9 as its substrate binding region, and the last three transmembrane regions were still unknown functional regions. The transmembrane region hPepT2 (579-664) of human oligopeptide transporter 10-11 was expressed in prokaryotic expression. The recombinant plasmid of pET30a () -hPepT2 (579-664) was constructed by designing primers for PCR cloning of cDNA of hPepT2, and then double restriction enzyme digestion and T4 ligase ligation were used to construct pET30a () -hPepT2 (579-664) recombinant plasmid. The recombinant plasmid was sequenced without amino acid mutation and then expressed in E.coli BL21 (DE3) pLysS. The expression of 0.3mM was induced by IPTG at 37 鈩

本文編號(hào)：2141680