人C5a新型受体（C5L2）N端突变体的功能研究

发布时间：2018-05-07 03:14

本文选题：C5a + C5L2　；参考：《第三军医大学》2007年硕士论文

【摘要】： 过敏毒素C5a是炎症反应的重要介质和趋化因子,在急性肺损伤、脓毒血症、脑膜炎、风湿性关节炎、肾小球肾炎等多种疾病的发生发展过程中起重要作用。C5a在体内被羧肽酶作用后,其C-端第74位精氨酸(Arg)迅速地分离,而成为去精C5a(C5a des-Arg~(74))。C5a通过其受体-CD88和C5L2(C5a like receptor-2)来实现生物学活性。C5a与CD88通过两点模型相互作用后,介导一系列的炎症反应,如激发细胞脱颗粒,释放组胺,增强血管通透性;诱导白细胞表达分泌IL-6等细胞因子。C5L2是C5a的新型受体,其最大的特点是与C5a/C5a des-Arg~(74)呈高亲和力结合,且不与G蛋白偶联、无信号传导的清道夫受体,它可能通过有效地结合过敏毒素(如:C5a/C3a/C4a等)在清除和降低炎症反应方面发挥主要作用。目前C5a-C5L2相互作用的结合位点和结合模型尚未揭晓,仅根据CD88的研究推测其也为两点结合模型,且C5L2 N端为主要的功能域,负责和绝大部分的配体结合。寻找C5L2蛋白和C5a的结合位点,可以全面了解其功能,同时通过作用于该位点激活或促进配受体的结合,理论上能够阻断或减少C5a-CD88的相互作用,从而降低C5a引起的炎症反应,为治疗炎性相关疾病找到一条新的通路。体外定点突变技术可以对某个区域内的特定碱基进行定点改变、缺失或插入,通过突变前后蛋白质功能的变化来研究其结构与功能的关系。本研究就是通过生物信息学分析C5L2与C5a的可疑结合位点,同源模建的方法预测C5L2三维立体结构;选取C5L2N端为研究对象,构建多个突变体,通过原核表达野生型和突变型蛋白,利用生物传感器检测突变前后C5L2 N端蛋白与C5a结合力的变化,从而初步定位与C5a发生相互作用的关键区域;再合成1条含有该位点的多肽,通过体外细胞实验和活体动物实验,验证该多肽拮抗炎症的效应,为进一步研究C5L2的功能以及治疗C5a相关疾病的研究奠定基础。实验方法 1.利用生物信息学技术分析C5L2结构特点及其分子的跨膜结构、二级结构、亲水性、可及性、柔韧性等;再通过同源模建的方法预测C5L2蛋白的三维立体结构。 2.利用基因重组技术和定点突变技术构建原核表达野生型和突变型C5L2-N端重组体;经IPTG诱导表达,亲和层析法纯化融合蛋白,最后经凝血酶Xa酶切纯化后,获得纯度较高的目的蛋白。 3.rhC5a包被于生物传感器芯片,分别检测原核表达的野生型和突变型蛋白与rhC5a的结合力。 4.合成1条含有结合位点的多肽,命名为D3,在体外预先与rhC5a孵育30min后,加入中性粒细胞培养体系,培养4小时后,其细胞上清液经ELISA检测IL-6水平;复制小鼠内毒素休克模型,通过对小鼠48h生存率的观察,初步研究该多肽在体内拮抗C5a的活性。实验结果 1.C5L2第3-22位、第179-183、第190-195位,第265-275位为其可能的配体结合域,由于C5L2 N-端,尤其是以ASP~(12,15,18)为中心的肽段呈现出高的亲水性、柔韧性及可及性,使其成为本次研究的对象。同源模建获得了C5L2蛋白的三维空间结构;表面静电势能图显示:C5a表面氨基酸残基富集正电荷,C5L2表面富集负电荷,二者可以通过正负电荷的吸引完成配受体的结合。在此基础上,以ASP为靶点,确定了5个突变位点,为进行下一步的分子生物学实验奠定了基础。 2.野生型表达载体经PCR和酶切方法初步鉴定构建成功,测序结果显示目的片段插入正确,并命名为W;突变型表达载体测序结果显示突变位点为设计位点,我们将突变成功的2个缺失突变体命名为D-1和D-2,3个点突变体分别命名为S-1,S-2和S-3。重组质粒转化成功后,IPTG诱导表达,获得了分子量约33 kDa的野生型及突变型融合蛋白;亲和层析技术获得纯度80%的融合蛋白;凝血酶Xa酶切纯化后,得到了约为4 kDa的目的蛋白。 3.rhC5a成功包被于生物传感器芯片。R4、W与S-1、S-2均可与rhC5a结合,结合率分别为88%,86%,85%和81%,四组问无显著性差异(P＞0.05);S-3也可与rhC5a结合,但结合率明显下降,为21%,与前四组蛋白结合率有显著性差异(P＜0.01);而D-1、D-2则不与rhC5a发生特异性结合反应,从而初步寻找到了与rhC5a结合位点密切相关的区域和关键的氨基酸残基。 4.多肽D3能显著减少rhC5a刺激后中性粒细胞产生IL-6(P＜0.01);D3对LPS所致内毒素休克小鼠具有明显的保护作用,使D3治疗组的生存率较对照组明显提高(P＜0.01)。结论 1.与CD88不同,C5L2 N-端ASP~(22,25,32)不是C5L2与C5a结合的关键氨基酸。 2.C5L2 N-端第10-21位氨基酸(NH2-YGDYSDLSDRPV-COOH)可能是C5L2与C5a结合的关键区域,其中ASP~(12,15,18)起关键作用。 3.得到了一条具有抗炎效应的多肽。
[Abstract]:C5a and CD88 interact with C5a / C5a des - Arg ~ ( 74 ) .

In vitro site - directed mutagenesis technique can be used to study the relationship between the structure and function of C5L2 in a certain region by site - directed mutagenesis , deletion or insertion . The study is to study the relationship between the structure and function of C5L2 by bioinformatics analysis .

experimental method

1 . The structure of C5L2 was analyzed by bioinformatics . The structure , secondary structure , hydrophilicity , accessibility , flexibility and so on were analyzed . The three - dimensional structure of C5L2 protein was predicted by homologous modeling .

2.Construction of prokaryotic expression wild - type and mutant C5L2 - N - terminal recombinant by using gene recombination technology and fixed - point mutation technology ; IPTG - induced expression and affinity chromatography to purify the fusion protein ; and finally , the target protein with higher purity is obtained after being cut and purified by thrombin Xa .

3 . rhC5a was coated on biosensor chip to detect the binding force of wild type and mutant protein expressed in prokaryotic expression and rhC5a respectively .

4 . A polypeptide containing binding site was synthesized and named D3 . After incubation with rhC5a in vitro for 30 min , neutrophil culture system was added . After 4 hours of culture , the supernatant of cells was detected by ELISA for IL - 6 levels . The mouse endotoxin shock model was replicated , and the activity of C5a in vivo was preliminarily studied by observing the 48h survival rate of mice .

experimental results

1 . C5L2 3 - 22 , 179 - 183 , 190 - 195 , 265 - 275 are their possible ligand binding domains . Since C5L2 N - terminal , especially the peptide segment centered on ASP ~ ( 12 , 15 , 18 ) exhibits high hydrophilicity , flexibility and accessibility , it becomes the object of this study .

2 . The wild type expression vector was constructed successfully by PCR and enzyme digestion method . The results showed that the target fragment was inserted correctly and named W . The mutant expression vector sequencing results showed that the mutation site was the design site . The two deletion mutants were named as D - 1 , S - 2 and S - 3 respectively . After the transformation of the recombinant plasmid , the fusion protein with molecular weight of about 33 kDa was obtained . The fusion protein with purity of 80 % was obtained by affinity chromatography .

3 . rhC5a was successfully coated on biosensor chip . The combination of R4 , W and S - 1 and S - 2 could be combined with rhC5a , the binding rate was 88 % , 86 % , 85 % and 81 % , respectively , but the binding rate was significantly decreased ( P < 0.01 ) .

4 . The IL - 6 ( P & lt ; 0.01 ) was significantly reduced by the polypeptide D3 after the stimulation of rhC5a , and D3 had obvious protective effect on LPS - induced endotoxic shock mice , and the survival rate of D3 treated group was significantly increased compared with the control group ( P < 0.01 ) .

Conclusion

1 . Unlike CD88 , C5L2 N - terminal ASP ~ ( 22 , 25 , 32 ) is not a key amino acid binding to C5L2 and C5a .

2.C5L2 N - terminal 10th - 21st amino acid ( NH2 - YGDYSDLSDRPV - COOH ) may be a key area of C5L2 binding to C5a , where ASP ~ ( 12 , 15 , 18 ) plays a key role .

3 . A polypeptide with anti - inflammatory effect is obtained .

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

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