【作者】 杨永涛；

【导师】 汪正清；

【作者基本信息】 第三军医大学 ， 微生物学， 2009， 硕士

【摘要】 补体系统的异常过度活化是缺血再灌注损伤、异种器官移植急性排斥反应等许多炎症性疾病的发生和发展的重要始动因素之一。血清补体抑制水平的降低会导致溶尿综合症、阵发性睡眠性血红蛋白尿、肾小球肾炎及遗传性血管源性水肿等疾病的发生。抑制补体的过度活化是治疗这些疾病的一个新的思路。补体活化有经典、旁路和甘露糖结合凝集素(MBL)三条途径,但这三条途径都在C3处汇合。补体1型受体(complement receptor type 1,CR1)能够结合C3b和C4b,具有加速C3/C5转化酶裂解的加速衰变活性(decay-accelerating activity DAA)和辅助I因子裂解C3b和C4b的辅助因子活性(co-factor activity CA)。由于CR1是一个多能有效的补体抑制剂,因此在CR1基础上研制补体抑制剂是一个十分合理的策略。由于CR1中的内在同源性,在除羧基端2个SCRs外的28个SCRs形成4个大的长同源重复(long homology repeat LHR),每个由7个SCRs组成。分别称为LHR A、LHR B、LHR C和LHR D。其中,LHR A中的前3个SCRs组成有功能性位点Ⅰ,而LHR B和LHR C中的前3个SCRs组成的位点只有三个氨基酸的不同,有相同的功能,都称为功能性位点Ⅱ。位点Ⅰ能够结合C4b,而结合C3b的能力非常微弱,对C3转化酶有很高的加速衰变活性,但辅助I因子裂解C3b和C4b的能力比较弱。位点Ⅱ能够有效地结合C3b和C4b,结合C4b的能力比结合C3b的能力弱。位点Ⅱ对于C3b和C4b有很高的CA活性,但对C3转化酶的DAA活性比较低,因此可以说一拷贝的加速衰变因子(decay-accelerating factor DAF)样位点和2个拷贝的膜辅助蛋白(membrane cofactor protein MCP;CD46)样位点使CR1成为补体活化调节剂(regulators of complement activation RCA)家族中唯一的能够失活裂解全部4种C3和C5转化酶的多功能性分子。试验表明,尽管LHR A对C3转化酶有足够的DAA活性,但对于C5转化酶的DAA活性较低,要使LHR A对C5转化酶具有较好的DAA活性,其后必须跟包含有位点Ⅱ的LHR B或LHR C。LHR B或LHR C的作用是结合三聚体的C5转化酶中的C3b。本研究从CR1分子的结构和功能出发,利用重叠延伸PCR(splicing overlap extention PCR,SOE-PCR)的方法构建出能够结合C3b和C4b的双功能域CR1衍生物,并在原核系统中进行表达并验证了其活性,为新型补体抑制剂的进一步研制奠定基础。研究取得了以下主要结果:1.从人外周血单个核细胞中提取总RNA,经RT-PCR成功地克隆了编码人补体受体Ⅰ型胞外区CR1-SCR1-3的cDNA(其长度为585bp),连接于pMD18-T simple载体后测序,序列比对发现与GenBank上登录的编码相应补体受体Ⅰ型cDNA区域序列一致。2.以构建的含功能性片段Ⅰ的T载体为模板,扩增出平端的功能性位点Ⅰ;以本室构建的pET32a-CR1-SCR15-18为模板扩增出平端的功能性片段Ⅱ(CR1-SCR15-17);利用重叠延伸PCR方法扩增出包含双功能域的融合基因。将融合基因重组于pET32a(+)载体,成功地构建了双功能域的重组原核表达质粒,命名为pET32- CR1-2D。氨苄青霉素筛选出阳性重组质粒经酶切及序列测定确认。3.将构建的pET32-CR1-2D载体成功转入大肠杆菌Rosetta(DE3),经IPTG诱导表达,SDS-PAGE分析发现在分子量约63kDa处出现一条明显表达条带;其以包涵体形式表达;并确立pET32- CR1-2D在37℃条件下,1mmol/L IPTG诱导4h时,大肠杆菌中有较高的蛋白表达水平。采用抗6×His抗体经Western blot进一步证实了此蛋白。4.蛋白经Ni-NTA柱纯化后,可得到单一条带的蛋白,表明采用亲和层析可实现Txr-CR1-2D蛋白的一步纯化。透析复性试验发现尿素浓度梯度逐步递减复性效果较好,最佳复性条件为:透析液中谷胱甘肽氧化型为1.5 mmol/L、还原型为3.5 mmol/L时析出蛋白较少,且补体溶血抑制实验表明此时生物活性较好;透析复性后的蛋白经肠激酶酶切,及随后Ni-NTA纯化后,可获得不含外源氨基酸的重组CR1-2D蛋白。5.体外功能试验验证了重组蛋白的活性。体外免疫介导红细胞溶解试验模型中,分光光度计方法测定上清中血红蛋白发现该蛋白能够抑制红细胞溶解;流式细胞术的方法发现重组蛋白能够减少红细胞表面C3b沉积;ELISA方法发现重组蛋白能够减轻上清中C5a产生。这些指标一定程度上说明了CR1-2D有抑制血管内溶血和血管外溶血的能力。在小鼠体内建立免疫介导红细胞溶解模型,流式细胞术的方法观察了输注红细胞在不同时相点存活情况。证实了该重组蛋白在标记红细胞输注的两小时内能够延长红细胞的存活时间。综上所述:本实验成功的构建了由位于LHR A内的功能性片段Ⅰ和位于LHR C内的功能性片段Ⅱ组成的双功能域的重组CR1分子,体内外功能试验验证了其生物学活性。我们的试验结果表明该重组双功能域蛋白研制开发新型补体抑制剂奠定了良好的基础。更多还原

【Abstract】 The complement system is a major component of the innate immune system, on the front line of the fight against infection, but itcan cause substantial cell and tissue damage when activated inappropriately. For this reason, activation of the complement system is under delicate regulation that involves a set of membrane and plasma inhibitors. A reduced level of component inhibition can result in a variety of diseases such as hemolytic uremic syndrome, paroxysmal nocturnal hemoglobinuria, glomerulonephritis,and hereditary angioedema, while overactivation of the complement cascade may be a contributing factor to ischemia reperfusion injury. Therefore, rational strategies are needed to design therapeutic agents that modulate complement activity, and are based on the mechanisms of the complement system’s natural inhibitors.The complement system can be activated by three pathways, known as the classical, alternative, and mannose-binding lectin pathways. All three converge at the point of cleavage of C3 into C3a and C3b. C3b then participates in the formation of C3 convertase in the alternative pathway, and C5 convertase. Thus, C3b is central to complement regulation. Complement receptor type 1 (CR1) is a complement activation family regulator, and is also known as the immune adherence receptor because it binds to C3b and C4b . CR1 can regulate the activation of the complement cascades by serving as a co-factor for factor I-mediated cleavage of C3b and C4b, and by accelerating the decay of both the classical and alternative pathway C3 and C5 convertases . For this reason, CR1 is the most potent and versatile complement inhibitor of the complement activation family . CR1 is composed of multiple of short consensus repeat (SCR) domains, a transmembrane domain and a small cytoplasmic domain, with each SCR containing about 60–70 amino acids. Based on degree of internal homology, all except the two carboxyl terminal SCRs can be classified into larger units, called long homologous repeats (LHR) A, B, C, and D, each of which is composed of seven SCRs. In CR1, three domains interact with C3b/C4b and with the convertases. One domain is located in LHR A, B, and C, more precisely in the first three SCRs of each LHR. The active domain in SCRs 1–3, called domain 1, is unique. The active domain in SCRs 8–10 is nearly identical to the domain in SCRs 15–17, with the exception of three amino acids. These are both called domain 2 . Structure and function relationship studies have demonstrated that domain 1 binds C4b and, very weakly, C3b. It has high decay-accelerating activity (DAA) for the C3 convertases, but low co-factor activity (CA) for factor I-dependent cleavage of C3b and C4b. Domain 2 binds C3b and C4b efficiently, with an affinity for C4b that is lower than for C3b, but comparable to the affinity for C4b of domain 1. It has high co-factor activity for both C3b and C4b, but low DAA for the C3 convertases . Efficient DAA for the C5 convertases by CR1 is achieved only if both domains 1 and 2 are present . Thus, combining the active sites within the N-terminal three SCRs of the LHR A and those of either LHR B or LHR C, could result in a smaller and more potent CR1-based inhibitor .In the present study, based on the potential complement inhibitory activities in the LHRs described above, we combined domain 1 of LHR A with domain 2 from LHR C using an overlap extension PCR method. We expressed the constructed CR1-based protein fused to thioredoxin in Escherichia coli, where it was mainly in inclusion bodies. After release and subsequent refolding, enterokinase cleavage, and purification, we verified its protective effects in an in vitro model of blood group alloimmune incompatibility, and in a mouse model of transfusion incompatibility. We demonstrated the CR1-based molecule could prolong red blood cells (RBC) survival in vivo, demonstrating its potential to be used as a potent and cost-effective complement inhibitor.The research mainly achieved the following results:1. RNA was isolated from fresh human peripheral blood mononuclear cells (PBMC) and the functional fragment1 was successfully amplified using the reverse transcription PCR method.The fragment was coloned into the pMD18-T simple plasmid.The sequencing result show the fragment sequence was completely agree with the corresponded sequence in the GeneBank.2.the blunt end fragment 1 was successfully amplifed using the above construct plasmid as template. The blunt end fragment 2 was succesfully amplfied using the plasmid PET32a-CR1-SCR15-18 that was constructed before. The fusion fragment was amplifed using the splicing overlap extention PCR(SOE-PCR) method,the fused frament was ligated into the PET32a plasmid. The constructed plasmid was verified by enzyme digestion and sequencing, The verified plasmid was named as PET32a-CR1-2D.3.the tansformed PET32a-CR1-2D/Rosetta(DE3) was induced by IPTG and analyzed by SDS-PAGE. Result show the fusion protein was highly expressed in E.coli in inclusion bodies form and the molecule mass of the expressed protein was 63 kD. Further research show the protein has the optimal expression level when induced for 4 hours at the concentration of 1mM IPTG under 37℃.western blot against 6xHis antibody further confirmed the expressed protein.4.The protein can be isolated by on-step affinity purification. The purity of the recombinat protein was up to 92% after Ni-NTA column affinity chromatography. We screened the dialysis refolding procedure and found the CR1-2D ptotein formed the least aggregates and had best bioactivity when refolded under the GSH and GSSG concentrations of 3.5 and1.5 mM, respectively. The gradual removal of the denaturing agent over time is essential in the refolding process. After clevege by enterokinase to remove the Trx Tag and subsequent Ni-NTA column affinity chromatography, we obtained the purified CR1-2D protein without extra amino acid.5.In vitro and in vivo functional experiments analysised the recombinant protein bioactivity. In the in vitro transfusion imcompatible model, Spectrophotometer measured the optical density of hemoglobin, result show the recombinant protein can inhibit hemolysis; flow cytometry method found the recombinant CR1-2D protein can reduce the deposition of C3b on the red blood cell membrane. Elisa method measured the C3a in the supernatant and found the recombinant protein can reduce C3a release. These results indicated the protein could inhibit hemolysis intravascularly and extravascularly to a certain extent. In mouse transfusion imcomptible model, flow cytometry counted the red blood cell marked by fluorescence dye, PKH67, at different time point, result show: for the initial 2 h post-transfusion, CR1-2D protein prolonged transfused RBC survival in the mouse circulation, compared to buffer-treated mice .In conclusion,we combined functional domain 1, located in the long homologous repeat (LHR) A, with functional domain 2,located in LHR C. We expressed the two-domain, two-function protein with an enterokinase site at the Nterminus and a termination codon at the C-terminus in Escherichia coli.We verified the bioactivity of the molecule in vitro and in vivo experiment model. Our results indicate that the CR1-based protein may be a model for developing smaller and more potent complement inhibitors for future therapeutics.更多还原