重组Exendin-4-胰高血糖素样肽1/IgG4(Fc)融合蛋白的表达和活性研究

目的  构建表达长效胰高血糖素样肽1（glucagon-like peptide 1,GLP-1）受体激动剂重组Exendin-4-GLP-1/IgG4(Fc)融合蛋白的质粒载体,并研究该融合蛋白的活性。方法  将编码Exendin-4-GLP-1/IgG4(Fc)的重组基因插入表达载体pOptiVEC™-TOPO^®来构建重组质粒Exendin-4-GLP-1/IgG4(Fc)-pOptiVEC™-TOPO^®。将构建的重组质粒转染CHO/DG44细胞并收获表达产物后,分别用亲和层析法和免疫印迹法对表达产物进行纯化和检测。通过胰岛素释放实验确认重组融合蛋白对INS-1细胞胰岛素分泌的影响,同时在CD1小鼠中研究该融合蛋白对血糖的调节作用。结果  转染重组质粒的CHO/DG44细胞可成功表达Exendin-4-GLP-1/IgG4(Fc)。蛋白质印迹法检测显示,纯化的表达产物的相对分子质量（M_r）与预期相符（重组融合蛋白单体和二聚体的M_r分别约为35 000和70 000）。胰岛素释放实验表明,在葡萄糖浓度恒定的情况下INS-1细胞分泌的胰岛素量随重组融合蛋白浓度的升高而增加。CD1小鼠实验显示,重组融合蛋白对链脲佐菌素诱导的糖尿病小鼠的血糖具有调节作用,Exendin-4-GLP-1/IgG4(Fc)处理的糖尿病小鼠的血糖明显低于对照糖尿病小鼠（F=3194,P＜0.01）。结论  Exendin-4-GLP-1/IgG4(Fc)具有天然GLP-1的活性,可作为GLP-1受体激动剂用于2型糖尿病的治疗。     

一种新穿膜肽的构建、表达及其活性检测

通过对穿膜肽TAT-PTD构效关系的分析和结构改造，构建能有效进入血脑屏障且低毒的新穿膜肽。该研究用分子克隆技术构建出表达型载体pET28a—T1-GFP，重组质粒在大肠杆菌Ecoli BL21（DE3）中表达融合蛋白His-T1-GFP，经组氨酸亲和层析纯化后，用荧光显微镜和免疫组化的方法来检测His—T1-GFP在活体动物小白鼠体内的跨膜递送作用。经测序证实成功构建了表达型载体pET28a—T1-GFP，融合蛋白His—T1-GFP在大肠杆菌EcoliB L21（DE3）中表达率为20％。经组氨酸亲和纯化，得到纯度达85％的融合蛋白。SDS-PAGE和Western Blotting显示纯化蛋白为His-T1-GFP，动物实验表明融合蛋白His—T1-GFP能够有效跨过血脑屏障，主要分布在大脑皮层和海马回。该研究成功地构建了-种新的穿膜肽-T1，为其携带有生物活性的大分子物质到达脑部进行蛋白治疗奠定了基础。     

以GLP-1受体为靶点的药物筛选模型的建立及功能鉴定

目的以GLP-1受体为靶点,建立GLP-1类似物活性检测的细胞模型,为GLP-1类似物以及GLP-1受体激动剂的药物筛选提供一种简单可靠的评价方法。方法首先将人源GLP-1受体基因插入pEGFP-N3,构建真核表达载体pEGFP-GLP-1R,并转染至HEK293A细胞中,经G418压力筛选后获得稳定表达GLP-1R-GFP的293A细胞株,然后通过GFP荧光信号和Western blot检测GLP-1R-GFP融合蛋白在该细胞中的表达与分布;最后利用GLP-1类似物利拉鲁肽刺激细胞,均相时间分辨荧光法检测细胞内cAMP含量变化。结果成功构建了GLP-1R-GFP-293A稳转细胞株,GLP-1RGFP蛋白主要分布在细胞膜表面,该细胞对GLP-1类似物利拉鲁肽具有高灵敏度和产生cAMP的特异性反应。结论利用所构建的细胞模型,可对小分子和GLP-1类似物进行体外活性分析,为筛选GLP-1受体激动剂奠定了模型基础。     

TAT-tCNTF融合蛋白对Aβ_(25-35)诱导SH-SY5Y细胞损伤的保护作用

目的确定TAT-tCNTF融合蛋白能够透过细胞膜进入细胞内,并研究其对Aβ诱导SH-SY5Y细胞损伤的保护作用。方法重组PCR获得人截短型CNTF基因后,与pBV220-TAT载体连接,在大肠杆菌中表达,细胞免疫荧光方法检测TAT蛋白运载融合蛋白穿过SH-SY5Y细胞膜的作用,Aβ25-35诱导SH-SY5Y细胞产生神经毒性,采用MTT(四甲基偶氮唑盐)法检测融合蛋白对细胞存活率的影响,Heochst33342/PI荧光双染法进行细胞凋亡和坏死的形态学观察,并进行LDH释放分析进一步检测TAT-tCNTF对细胞损伤后的保护作用。结果成功构建pBV220-TAT-tCNTF表达载体,Western blot结果表明重组融合蛋白可以与CNTF抗体特异性结合,细胞免疫荧光方法显示TAT-tCNTF能大量进入细胞,而rhCNTF只有微量进入细胞。对Aβ25-35诱导损伤的SH-SY5Y细胞MTT、Heochst33342/PI荧光双染法及LDH分析都表明TAT-tCNTF能明显提高细胞的存活率。结论TAT-tCNTF融合蛋白可以跨越细胞膜,能够保护Aβ25-35诱导损伤的SH-SY5Y细胞,具有促进细胞生存生长的活性。     

pET15b-pep-1-p27mt的构建、表达及意义

构建细胞穿膜肽(cell-penetrating peptides,CPPs)PEP-1和细胞周期抑制蛋白p27mt的重组体,并在大肠杆菌中表达。将已构建的表达细胞周期抑制蛋白p27mt的基因,克隆入pET15b-pep-1原核表达载体,在大肠杆菌BL21(DE3)LysS内,以IPTG诱导融合蛋白表达。表达产物用SDS-PAGE及Western blot分析鉴定。结果:成功地构建PEP-1-p27mt融合蛋白原核表达载体,并在IPTG诱导下获得特异性的表达。PEP-1-p27mt融合蛋白表达载体的构建,为体内应用细胞周期抑制蛋白诱导肿瘤细胞凋亡提供了理论基础。     

增强型绿色荧光蛋白与IgG抗体亲和肽融合蛋白的制备

目的构建、表达增强型绿色荧光蛋白(EGFP)与IgG抗体亲和肽融合蛋白,并对其生物学功能进行研究。方法将EGFP基因与IgG抗体亲和肽基因重组,构建原核分泌表达载体pSpA-EGFP-His,通过竞争ELISA和荧光光谱测定,考察其在大肠杆菌中表达产物SpA-EGFP融合蛋白的生物学活性。结果SpA-EG-FP融合蛋白的相对分子质量42kD,与理论值相近,荧光光谱与文献报道一致,且能与SpA-Peroxidase竞争结合反应体系中的兔IgG抗体。结论SpA-EGFP融合蛋白在E.coliDH5α中正确表达,且具有EGFP的荧光特性和与哺乳动物IgG抗体结合的生物学活性。     

人心肌肌钙蛋白I(cTnI)基因在大肠杆菌中的融合表达和纯化

目的:研究人cTnI基因在大肠杆菌中的融合表达和纯化.方法:以pCOMb3-cTnI为模板,用PCR方法得到了编码该蛋白的基因,进一步将该基因克隆到中间载体pUCm-T,再酶切得到cTnI基因插入到pKL载体中,构建成表达质粒pKL-cTnI.在BL21细胞中经IPTG诱导表达,利用载体携带6×His纯化标签,以HisTrap Kit纯化重组蛋白,并进行免疫活性测定.结果:融合蛋白在大肠杆菌中得到高效表达,并用HisTrapKit成功纯化目的蛋白.ELISA分析证实该重组蛋白具有与天然心肌肌钙蛋白相似的免疫活性.结论:本研究为建立急性心肌梗死早期诊断试剂奠定了基础.     

抗菌抗癌肽CM4N在大肠杆菌中的融合表达与活性鉴定

为探讨内含肽intein在抗菌肽表达与纯化中的应用,采用递归PCR法合成CM4末端添加Asn的基因CM4N,克隆至E.coli表达载体pTYB11中,构建了与intein的C端融合的表达载体pTYYB11-CM4N.重组质粒转化至E.coli BL21(DE3)中进行IPTG诱导表达,融合蛋白intein-CM4N主要以可溶形式存在于胞内.利用载体中intein中的chitin结合域,将融合蛋白通过chitin亲和层析一步纯化,经DTT诱导intein的自我切割活性,实现CM4N在亲和柱上的切割与分离,透析冻干后得到了纯度95%以上的重组CM4N.活性检测结果显示,重组CM4N具有抗大肠杆菌、沙门氏菌和K562肿瘤细胞的活性.研究认为,内含肽在抗菌肽的基因工程中可能具有重要的应用前景.     

胞苷磷酸激酶基因的克隆与原核表达

目的:克隆胞苷磷酸激酶(CMPK)基因,构建携带CMPK基因的原核表达载体,诱导表达具有活性的重组CMPK融合蛋白,获得转基因工程菌.方法:利用PCR法扩增大肠杆菌基因组DNA,纯化的PCR产物链接至pMD18-T载体上,得到重组质粒pMD18-T-CMPK,转化E-coli BL21(DE3)感受态细胞,酶切鉴定;分别用BamH I和XhoI限制性内切酶双酶切重组质粒pMD18-TCMPK和pET28a(+)表达载体.连接后,转入E.coli BL21(DE3)感受态细胞,酶切鉴定.用终浓度1 mmol/L的IPTG诱导一定时间(3、6h)后,取E.coli上清液做电泳分析.结果:所获CMPK基因全长为786 bp,编码含有262个氨基酸的蛋白,当A值为0.6～0.8时重组质粒经IPTG诱导3h后,相对分子质量约30000处出现目的蛋白条带,随着时间的延长,蛋白表达量增加不明显.结论:成功地克隆CMPK基因,表达了大肠杆菌BL21(DE3)重组CMPK融合蛋白,为胞苷磷酸激酶进一步开发和应用奠定基础.     

2种Heymann肾炎受体相关蛋白融合蛋白的制备及其意义

目的:构建Heymann肾炎致病原受体相关蛋白(RAP)全长和羧基端多肽的原核融合表达载体,表达并纯化相应的融合蛋白.方法:通过RT-PCR法获得RAP全长的cDNA,将其与pGEM-T克隆载体连接并转化,对阳性克隆进行测序分析,设计针对RAP全长和羧基端带有EcoR Ⅰ和Xho Ⅰ双酶切位点的特异引物,通过PCR合成RAP全长359个氨基酸和羧基端108个氨基酸的DNA.纯化后与含有GST的pGEX-4T-1载体体外重组连接,亚克隆到感受态细胞BL21中,经IPTG诱导,表达的融合蛋白通过GST-Sepharose 4B亲和层析法纯化,并经SDS-PAGE和Western Blot检测.结果:酶切分析、PCR及DNA序列测定结果表明成功构建了重组原核表达载体pGEX-4T-1-RAP1083和pGEX-4T-1-RAP324,含有重组质粒的大肠杆菌BL21经诱导后表达出RAP全长70 ku和羧基端40 ku的融合蛋白.Western Blot结果显示分别在分子质量70ku、40ku处出现特异性条带,与理论相符.结论:成功构建了RAP1083、RAP324与pGEX-4T-1融合基因表达载体,融合蛋白在大肠杆菌中高效表达,为后续研究Heymann肾炎的分子发病机制奠定了基础.     

sCR1在原核中表达、纯化、复性及活性鉴定

目的采用原核表达载体pET28a在E.coli BL21(DE3)中获得高表达、高活性的重组人sCR1融合蛋白。方法从人外周血中提取总RNA,应用RT-PCR获得人sCR1全长cDNA,将其克隆入原核表达载体pET28a中,构建含人sCR1的重组质粒(pET28a-sCR1),经测序鉴定正确,转化入E.coli BL21(DE3)中,经IPTG诱导,表达产物经SDS-PAGE分析和Western blot鉴定,通过Ni2+-NTA agarose亲和层析纯化后进行复性及生物学活性鉴定。结果获得原核表达载体pET28a-sCR1,经PCR鉴定及测序鉴定,得到重组E.coli BL21(DE3)克隆菌株,经IPTG诱导含有pET28a-sCR1的E.coli BL21(DE3)克隆菌,表达出重组人sCR1融合蛋白。此蛋白在SDS-PAGE上表现为Mr大于29000的蛋白区带,在Western blot分析中可被sCR1的CD35单克隆单抗体(mAb)识别。经Ni2+-NTA agarose亲和层析纯化、复性后得到高纯度的sCR1融合蛋白表达及较高的生物学活性。结论人sCR1融合蛋白在E.coli BL21(DE3)表达系统中的高水平表达,并且有与人体天然蛋白相同的抗原性及其生物学活性。     

异源性Th表位修饰的CTLA4-Ig—msBlys构建及融合蛋白表达

目的构建异源性Th表位修饰的CTLA4-Ig-msBlys的原核表达质粒,并在EcoliBL21中诱导融合蛋白表达,初步检测蛋白特性。方法采用PCR法从质粒pCMV-sBlys扩增sBlys片段,将与卵清蛋白（ovalbumin,OVA）Th表位重组后的msBlys连接CTLA4-Ig片段,克隆入pGEX-KG原核表达载体,用LB固体培养基抗生素筛选、酶切并经琼脂糖凝胶电泳鉴定;将质粒pGEX—KG-CTI。A4-Ig—msBlys转入E．coliBL21菌株中,实现插入基因的融合表达,用SDS-PAGE和Western-blot检测表达产物。结果pGEX—KG—GTLA4-Ig—msBlys构建正确且最终转人E．coliBL21,得到融合蛋白的表达;CTLA4-Ig-msBlys融合蛋白在E．coli中获得表达,表达产物的相对分子质量同预期值一致。结论成功构建原核表达质粒pGEX-KG-CTLA4-Ig-msBlys,并在E．coliBL21表达CTLA4-Ig-msBlys融合蛋白,为下一步探讨Blys在自身免疫性疾病患者的治疗作用奠定了基础。     

肺癌靶向血栓蛋白hu3D3V_H-tTF的表达及活性鉴定

目的制备一种用于肺癌血管靶向栓塞治疗的融合蛋白hu3D3VH-tTF,并鉴定其生物学活性。方法利用重叠PCR技术构建tTF与hu3D3VH的融合基因,克隆至表达载体pET22 b(+),在E.coliBL21(DE3)中表达,镍亲和色谱柱纯化目的蛋白。ELISA检测融合蛋白hu3D3VH组分与肺腺癌细胞A549选择性结合活性,凝血实验和FⅩ活化实验鉴定融合蛋白tTF组分的促凝血活性。结果获得序列正确的hu3D3VH/tTF/pET22 b(+)重组子,融合蛋白在E.coliBL21(DE3)中高效表达。纯化后的融合蛋白与肺腺癌细胞A549具有选择性结合活性,并能活化FⅩ、有效促发血液凝固。结论成功构建hu3D3VH/tTF/pET22 b(+)重组子,hu3D3VH/tTF融合蛋白具有hu3D3VH的选择性结合能力同时具有TF的促凝血活性,为开展选择性肺肿瘤血管血栓性栓塞研究奠定了基础。     

The major determinant of exendin-4/glucagon-like peptide 1 differential affinity at the rat glucagon-like peptide 1 receptor N-terminal domain is a hydrogen bond from SER-32 of exendin-4

BACKGROUND AND PURPOSE

Exendin-4 (exenatide, Ex4) is a high-affinity peptide agonist at the glucagon-like peptide-1 receptor (GLP-1R), which has been approved as a treatment for type 2 diabetes. Part of the drug/hormone binding site was described in the crystal structures of both GLP-1 and Ex4 bound to the isolated N-terminal domain (NTD) of GLP-1R. However, these structures do not account for the large difference in affinity between GLP-1 and Ex4 at this isolated domain, or for the published role of the C-terminal extension of Ex4. Our aim was to clarify the pharmacology of GLP-1R in the context of these new structural data.

EXPERIMENTAL APPROACH

The affinities of GLP-1, Ex4 and various analogues were measured at human and rat GLP-1R (hGLP-1R and rGLP-1R, respectively) and various receptor variants. Molecular dynamics coupled with in silico mutagenesis were used to model and interpret the data.

KEY RESULTS

The membrane-tethered NTD of hGLP-1R displayed similar affinity for GLP-1 and Ex4 in sharp contrast to previous studies using the soluble isolated domain. The selectivity at rGLP-1R for Ex4(9–39) over Ex4(9–30) was due to Ser-32 in the ligand. While this selectivity was not observed at hGLP-1R, it was regained when Glu-68 of hGLP-1R was mutated to Asp.

CONCLUSIONS AND IMPLICATIONS

GLP-1 and Ex4 bind to the NTD of hGLP-1R with similar affinity. A hydrogen bond between Ser32 of Ex4 and Asp-68 of rGLP-1R, which is not formed with Glu-68 of hGLP-1R, is responsible for the improved affinity of Ex4 at the rat receptor.     

DPP4 deficiency preserves cardiac function via GLP-1 signaling in rats subjected to myocardial ischemia/reperfusion

Dipeptidyl peptidase-4 (DPP4) enzyme inhibition has been reported to increase plasma glucagon-like peptide-1 (GLP-1) level for controlling postprandial glucose concentration. Both DPP4 inhibitors and GLP-1 analog have been approved for antihyperglycemic agents. In addition to the insulinotropic effect, GLP-1 signaling was reported to modulate cardiac function. DPP4 inhibition was shown to improve survival rate after myocardial infarction in mice, but the precise mechanism remains unknown. We aimed to compare the cardiovascular responses of ischemia/reperfusion (I/R) between wild-type and DPP4-deficient rats and investigate the underlying mechanism. Rats were subjected to 45 min of coronary artery occlusion, followed by reperfusion for 2 h. Cardiac function was characterized by analyzing pressure–volume loops. As compared to wild-type rats, after I/R, DPP4-deficient rats had better cardiac performance in association with less infarct size and cardiac injury markers (LDH, ANP, and BNP), which could be attenuated by exendin-(9–39), a GLP-1 receptor antagonist. Exendin-(9–39) could diminish the increased phosphorylation levels of myocardial AKT and GSK-3β as well as the higher expression of GLUT4 in post-infarcted DPP4-deficient rats. However, exendin-(9–39) could not completely abrogate the less infarct size in DPP4-deficient rats as compared with that in wild-type rats, implicating the involvement of GLP-1 receptor-independent pathway. In summary, this study demonstrated that the benefit of cardiac protective action against I/R injury was demonstrated in DPP4-deficient rats, which is mediated through both GLP-1 receptor-dependent and receptor-independent mechanisms.     

Exendin-4 reduces glycemia by increasing liver glucokinase activity: an insulin independent effect

Exendin-4 is a stable peptide agonist of GLP-1 receptor that exhibits insulinotropic actions. Some in vivo studies indicated insulin-independent glucoregulatory actions of exendin-4. That finding prompted us to evaluate effects of exendin-4 on liver glucose metabolism. Acute and chronic treatment of exendin-4 resulted in increased hepatic glucokinase activity in db/db mice but not in lean C57 mice. The stimulatory effect of exendin-4 on glucokinase activity was abrogated by exendin 9-39, a GLP-1 antagonist. Exposure of hepatocytes isolated from db/db mice to exendin-4 elicited a rapid increase in cAMP, which was synergized by IBMX, an inhibitor of cAMP degradation. The GLP-1 antagonist, exendin 9-39, has abolished the cAMP generating effects of exendin-4 as well. Furthermore, chronic treatment of exendin-4 in streptozotocin-treated C57 mice resulted in restoration of hepatic glycogen, an indicator of improved glucose metabolism, without apparent changes in serum insulin levels. In conclusion, exendin-4 increased glucokinase enzyme protein and activity in liver via a mechanism parallel to and independent of insulin. Exendin-4-induced increase in hepatic glucokinase activity is more pronounced in the presence of hepatic insulin resistance. This beneficial effect of exendin-4 on liver glucokinase activity may be mediated by GLP-1 receptor.     

Exendin-4 protects the hearts of rats from ischaemia/reperfusion injury by boosting antioxidant levels and inhibition of JNK/p66Shc/NADPH axis

Exendin-4, a glucagon-like peptide-1 receptor agonist, was shown to protect against cardiac ischaemia/reperfusion (I/R) injury by suppressing oxidative stress. p⁶⁶Shc, a pro-oxidant and an apoptotic protein, is activated in the infarcted left ventricles (LVs) after induction of I/R. This study investigated if the cardiac protective effect of Exendin-4 against I/R injury in rats involves inhibition of p⁶⁶Shc and to determine the underlying mechanisms behind this. Adult male rats (n = 12/group) were divided into four groups as a sham, a sham + Exendin-4, an I/R, and an I/R + Exendin-4. Exendin-4 was administered to rats 7 days before the induction of I/R. Ischaemia was induced by ligating the left anterior descending (LAD) coronary artery for 40 minutes followed by reperfusion for 10 minutes. The infarct myocardium was used for further analysis. Exendin-4 significantly reduced infarct area (by 62%), preserved LV function and lowered serum levels of LDH and CK-MB in I/R-induced rats. Also, it significantly reduced LV levels of ROS and MDA and protein levels of cytochrome-c and cleaved caspase-3 but significantly increased levels of glutathione (GSH) and manganese superoxide dismutase (MnSOD) in LVs of I/R rats indicating antioxidant and anti-apoptotic effects. Furthermore, it inhibited JNK and p⁶⁶Shc activation and downregulated protein levels of p⁶⁶Shc and NADPH oxidase with no effect on protein levels/activity of p53 and PKCβII. Of note, Exendin-4 also increased GSH and MnSOD in LVs of control rats. In conclusion, Exendin-4 cardioprotective effect in I/R hearts is mediated mainly by antioxidant effect and inhibition of JNK/P⁶⁶Shc/NADPH oxidase.     

大鼠谷氨酸受体2抗体制备及其与Caspase 3相互作用分析

目的克隆大鼠谷氨酸受体2(GluR2)基因片段,构建重组表达质粒,在原核系统诱导表达,制备多克隆抗体,分析GluR2与caspase3的相互作用。方法从大鼠脑组织提取总RNA,实时聚合酶链反应(RT-PCR)扩增GluR2基因抗原性较强的一段,定向克隆入表达载体pGEX-4T1,构建GST-GluR2融合蛋白表达质粒,转化至大肠杆菌BL21,经IPTG诱导表达GluR2蛋白。用SDS-PAGE方法分离目的蛋白,免疫家兔,制备抗血清。用Western Blot方法鉴定抗体的特异性,用Co-IP实验分析GluR2和caspase3的相互作用。结果SDS-PAGE显示GST-GluR2融合蛋白诱导表达成功;Western Blot显示制备的多克隆抗体具有较高的特异性;Co-IP显示大鼠脑组织中GluR2和caspase3存在相互作用。结论成功获得兔抗鼠GluR2特异性抗血清;脑组织中GluR2和caspase3存在相互作用,为进一步研究神经毒作用导致GluR2下调的分子机制奠定基础。     

抗AFP重链可变区单域抗体融合蛋白的构建、表达及初步鉴定

目的 构建抗甲胎蛋白(AFP)重链可变区(VH)单域抗体融合蛋白基因在大肠杆菌中的表达,并初步鉴定表达产物的活性.方法 从已构建的抗AFP单链抗体(ScFv)的载体中,经PCR扩增出VH 单域抗体基因,再克隆到融合蛋白表达载体pET32a( )中进行表达;用SDS-PAGE及Western-blot鉴定表达产物;并通过竞争抑制ELISA分析TrxA-VH融合蛋白的结合活性;细胞免疫组化染色分析其内化及结合情况.结果 VH 单域抗体基因全长339 bp,将其克隆到pET32a( )中,转化大肠杆菌可获得高效表达,Western-blot证实在相应分子质量处,有TrxA-VH融合蛋白的显色条带.经初步纯化和复性后,获得抗AFP的VH单域抗体融合蛋白.竞争抑制ELISA及细胞免疫组化证明,表达产物具有与AFP特异结合的活性.结论 成功构建了抗AFP的VH单域抗体融合蛋白,为临床的应用研究奠定了基础.