PACAP及其衍生物的克

发布时间：2018-05-07 08:55

本文选题：垂体腺苷酸环化酶激活多肽 + 饮食抑制　；参考：《华东师范大学》2011年硕士论文

【摘要】：垂体腺苷酸环化酶激活多肽(pituitary adenylate cyclase-activating polypeptide, PACAP)是血管活性肠肽(vasoactive intestinal polypeptide, VIP)/促胰液腺素/胰高血糖素/生长激素释放激素家族的一员,与VIP有68%的同源性。同VIP一样,PACAP是一种多效性多肽,其生物学功能涉及机体9大系统,而且对糖尿病、肥胖、高血压、哮喘、帕金森等疾病的治疗有一定的积极作用。然而,PACAP的体内半衰期较短(约5-10min),且目前针对这一问题的研究较少,并都需要进行体外化学修饰,操作繁琐、所得衍生物活性受限。因此,本研究对PACAP的氨基酸序列进行改造,在其羧基端末尾添加半胱氨酸,拟使其能与机体血清白蛋白结合,抑制可能存在的降解行为,提高体内稳定性；并用基因工程技术制备PACAP及其衍生物,降低研究成本；此外,本研究还通过动物实验和细胞实验初步探究了PACAP及其衍生物在抗肥胖、抗肿瘤和降血糖方面的活性。本研究的主要内容和结果概述如下： 1) PACAP及其衍生物的克隆、表达、纯化对GenPept报道的PACAP(包括PACAP38和PACAP27)的氨基酸序列进行改造,将改造衍生物命名为cPACAP (cPACAP38和cPACAP27);人工合成cPACAP38的碱基序列后,PCR扩增PACAP38、PACAP27和cPACAP27的基因序列；为使目的多肽与硫氧还蛋白(Trx)融合表达,采用克隆技术将目的基因插入表达载体pET32a(+)中,获得4种表达质粒后分别转化表达宿主菌BL21(DE3),得到4种表达菌株——BL21-pET32a(+)-PACAP38、BL21-pET32a(+)-cPACAP38、BL21-pET32a(+)-PACAP27、BL21-pET32a(+)-cPACAP27。表达菌株经IPTG诱导后表达融合蛋白,共4种——Trx-PACAP38、Trx-cPACAP38、Trx-PACAP27、Trx-cPACAP27,其表达量分别为22.1%、24.6%、47.3%、47.7%,可溶性融合蛋白占表达量的百分比分别为48.7%、54.0%、88.3%、66.6%。将IPTG诱导表达的菌液超声破碎后,经两次镍离子螯合亲和层析,得到4种目的多肽——PAC AP38、cPAC AP38、PAC AP27、cPAC AP27,得量分别为1.6mg、1.8mg、9.1mg、5.7mg, ImageJ软件扫描纯度均在95%以上。 2) PACAP及其衍生物的活性测定与比较采用小鼠饮食抑制实验,对基因工程制备的PACAP及其衍生物进行活性测定和比较。结果表明,不同剂量的4种目的多肽对小鼠饮食均有抑制作用,而且cPACAP38与cPACAP27的活性明显比PACAP38与PACAP27强,cPACAP38的效果能持续12h仍有显著性差异,cPACAP27也能维持12h有统计意义。表明与天然PACAP相比,衍生物cPACAP的体内活性更稳定、效果更持久。 3) PACAP及其衍生物生物学活性探究长期给药观察PACAP38及cPACAP38的抗肥胖效果。连续10天给小鼠腹腔注射PACAP38和cPACAP38,结果显示,小鼠体重、糖耐量及体内肥胖相关指标如甘油三酯、高密度脂蛋白、低密度脂蛋白和胆固醇等并没有明显变化。用MTT比色分析法检测不同浓度PACAP27和cPACAP27对人乳腺癌细胞Bcap37、人肝癌细胞BEL7404、人神经母细胞瘤细胞SH-SY5Y、人肺癌细胞A549增殖活性的影响,结果未显示出调控作用。用腹腔糖耐量及降糖药效法分析PACAP27和cPACAP27对GLP-1降糖活性的影响。发现,将PACAP27及cPACAP27与GLP-1混合后给小鼠注射,其降糖效果更明显,给药剂量为1 Onmol/kg时,混合给药组的降糖效果能持续1.5h,是单独GLP-1给药组的3倍,提示PACAP对GLP-1的降糖活性有辅助作用。通过以上研究,得到4种基因工程表达菌株,获得了体内生物学活性更稳定、效果更持久的PACAP衍生物(cPACAP38和cPACAP27),并通过小鼠体内实验证明了PACAP对GLP-1降血糖效果的辅助作用,为PACAP的进一步理论和应用研究奠定基础。
[Abstract]:As VIP , PACAP is a kind of multipotent polypeptide which has a positive effect on the treatment of diabetes , obesity , hypertension , asthma and Parkinson ' s disease .
PACAP and its derivatives were prepared by genetic engineering technique , and the research cost was reduced .
In addition , the activities of PACAP and its derivatives in anti - obesity , anti - tumor and blood glucose lowering were investigated by animal experiments and cell experiments .

The main contents and results of this study are summarized below :

1 ) Cloning , expression and purification of PACAP and its derivatives

The amino acid sequences of PACAP ( including PACAP 38 and PACAP 27 ) reported by GenPept were modified . The modified derivatives were named cPACAP ( cPACAP 38 and cPACAP27 ) . After the nucleotide sequence of cPACAP38 was synthesized , PACAP 38 , PACAP27 and cPACAP27 were amplified by PCR .
The expression vector pET32a ( + ) was inserted into the expression vector pET32a ( + ) , and the expression vector pET32a ( DE3 ) was transformed into BL21 ( DE3 ) . The expression vector pET32a ( + ) - cPACAP38 , BL21 - pET32a ( + ) - PACAP27 , BL21 - pET32a ( + ) - cPACAP27 were obtained .

The expression of soluble fusion protein was 48.7 % , 54.0 % , 88.3 % and 66.6 % , respectively .

Four kinds of polypeptide _ PAC AP38 , cPAC AP38 , PAC AP27 and cPAC AP27 were obtained after ultrasonic disruption of the bacterial solution induced by IPTG . The yield of PAC AP38 , PAC AP27 , cPAC AP27 was 1.6 mg , 1.8 mg , 9.1 mg , 5.7 mg , respectively , and the scanning purity of ImageJ software was above 95 % .

2 ) Determination and Comparison of the Activity of PACAP and Its Derivatives

The results showed that the activity of cPACAP 38 and cPACAP 27 was significantly higher than that of PACAP 38 and PACAP 27 , and cPACAP 27 could be maintained for 12 h .

3 ) Biological activity of PACAP and its derivatives

PACAP38 and cPACAP38 were injected intraperitoneally for 10 days . The results showed that the indexes such as triglyceride , high density lipoprotein , low density lipoprotein and cholesterol did not change in mice .

The effects of PACAP27 and cPACAP27 on proliferation activity of human breast cancer cell line Bcap37 , human hepatoma cell BEL7404 , human neuroblastomas SH - SY5 and human lung cancer cell A549 were detected by MTT colorimetric assay .

The effects of PACAP 27 and cPACAP27 on GLP - 1 hypoglycemic activity were analyzed by intraperitoneal glucose tolerance and hypoglycemic drugs . It was found that PACAP 27 and cPACAP27 were injected into mice after being mixed with GLP - 1 . The results showed that the hypoglycemic effect of PACAP 27 and cPACAP27 was 1 . 5h , which was 3 times higher than that of GLP - 1 alone , suggesting that PACAP had an auxiliary effect on the hypoglycemic activity of GLP - 1 .

The PACAP derivatives ( cPACAP38 and cPACAP27 ) were obtained in four kinds of gene engineering strains . The results showed that PACAP derivatives ( cPACAP38 and cPACAP27 ) were more stable and more effective in vivo , and the effect of PACAP on GLP - 1 hypoglycemic effect was proved through in vivo experiments , which laid the foundation for further theoretical and application research of PACAP .

【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R346

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