大肠杆菌中高效表达rhBMP-4的探讨及初步活性测定

目的 在大肠杆菌中表达具有生物活性的rhBMP-4。方法 在不改变氨基酸序列的前提下,以全基因合成的方式对人BMP-4成熟肽基因全长进行定点突变,将之重组入pET-3c表达载体并转化至大肠杆菌BL21(DE)plysS。IPTG诱导和包涵体复性后,利用C2C12细胞横向成骨细胞分化实验以及小鼠肌袋异位骨形成实验检测其活性。 结果 获得0.348 kb的BMP-4 DNA序列,表达的目的蛋白主要以包涵体的形式存在。经纯化及复性后,体内与体外的活性检测表明rhBMP-4有良好的诱骨生成活性。结论 该方案能够实现rhBMP-4在大肠杆菌中的高效表达。     

重组人骨形成蛋白-3成熟肽的表达、纯化及诱骨活性的研究*

推测人骨形成蛋白-3羧基端的127氨基酸的肽段为其成熟肽(hRMP 3m)。将编码此成熟肽的cDNA插入含PL启动子的表达质粒pDH中,构建表达质粒pDH-B-3m,转化大肠杆菌DH5α。42℃热诱导6h后表达量达到最高水平,约占菌体总蛋白28％;表达产物以包涵体形式存在。包涵体经分离和洗涤后．溶解于尿素,在变性溶解状态下经阳离子交换层析,目的蛋白纯度至少在95％以上。再经稀释复性。然后将纯化、复性的重组人骨形成蛋白3成熟肽(rhBMP－3m)植入小鼠肌肉间隙,于不同时间取材观察,在局部可见典型的软骨形成、软骨内成骨以及骨组织形成的过程,证实所制备的rhBMP－3m具有明显的异位诱骨活性。     

重组人骨形成蛋白—3成熟肽的表达,纯化及诱骨活性的研究

推测人骨形成蛋白３羧基端的１２７氨基酸的肽段为其成熟肽（ｈＢＭＰ３ｍ）。将编码此成熟肽的ｃＤＮＡ插入含ＰＬ启动子的表达质粒ｐＤＨ中,构建表达质粒ｐＤＨＢ３ｍ,转化大肠杆菌ＤＨ５α。４２℃热诱导６ｈ后表达量达到最高水平,约占菌体总蛋白２８％;表达产物以包涵体形式存在。包涵体经分离和洗涤后,溶解于尿素,在变性溶解状态下经阳离子交换层析,目的蛋白纯度至少在９５％以上。再经稀释复性。然后将纯化、复性的重组人骨形成蛋白３成熟肽（ｒｈＢＭＰ３ｍ）植入小鼠肌肉间隙,于不同时间取材观察,在局部可见典型的软骨形成、软骨内成骨以及骨组织形成的过程,证实所制备的ｒｈＢＭＰ３ｍ具有明显的异位诱骨活性。     

骨形成蛋白10成熟肽在大肠杆菌中的表达纯化及活性分析

目的:以生物制备骨形成蛋白10(BMP10)为目标,研究BMP10成熟肽在大肠杆菌中的表达及活性。方法:以人源BMP10成熟肽基因为模板,PCR获得N端带有组氨酸标签(6×His)的融合基因6×His-m BMP10,构建p ET28a/m BMP10表达载体;热击转染大肠杆菌BL21(DE3)菌株,卡那霉素抗性筛选获得重组表达菌株BL21/p ET28a-6×His-m BMP10,IPTG诱导表达后利用SDS-PAGE电泳、Western印迹对蛋白进行分析;超声波破碎菌体,收集包涵体,镍柱亲和层析纯化获得电泳纯目的蛋白;透析复性后,非还原SDS-PAGE检验目标蛋白的二聚体形成;通过体外细胞实验检测蛋白活性。结果:纯化得到纯度90%以上的m BMP10,复性后二聚体得率约为40%;活性实验测得P19细胞的Smad6蛋白表达上调3倍左右。结论:通过大肠杆菌表达体系获得具有生物活性的BMP10,为后续作用机理研究奠定了实验基础。     

苏云金杆菌vip3A基因的克隆、表达及杀虫活性分析

用全长PCR方法从野生型苏云金杆菌（Bacillus thuringiensis,Bt）菌株S184中克隆了2.3 kb大小的vip3A基因并进行了序列分析。将vip3AS184基因插入表达载体pQE30构建了表达质粒pOTP,转化大肠杆菌M15,转化子经1mmol/L IPTG诱导后可表达89 kD大小的Vip3A-S184蛋白,并得到Western blot证实。蛋白可溶性试验表明,目的蛋白中约有19%是可溶的,用透射电镜观察到大多数蛋白是以包涵体形式存在的。因此,可以在自然条件下进行目的蛋白的纯化和对家兔进行免疫制备多克隆抗体,用于苏云金杆菌Vip3A蛋白表达的检测。利用IPTG进行诱导培养的菌液对甜菜夜蛾（Spodoptera exigua）、斜纹夜蛾(S.litura)和棉铃虫(Helicoverpa armigera)等3种害虫的初孵幼虫进行生物测定,结果表明,Vip3AS184蛋白对夜蛾科害虫具有较高的杀虫活性。     

ProNGF在大肠杆菌中的表达、纯化和复性

将含有前导肽的人神经生长因子基因(proNGF)克隆在原核表达载体pET15b中, 转化大肠杆菌BL21(DE3)pLysS, 经IPTG诱导实现了目标融合蛋白的高效表达。SDS-PAGE分析表明表达蛋白占全菌总蛋白的20%左右, 表达蛋白主要以包涵体的形式存在。用6 mol/L的盐酸胍溶解包涵体后, 通过Ni2+-NTA柱纯化, 获得纯化的目标融合蛋白, 电泳谱带扫描分析表明蛋白纯度可达90%以上。Western blotting检测显示, 表达产物有较强的免疫学活性。经肠激酶作用后得到proNGF非融合蛋白, 分子量为27 kD, 100 mL表达菌液可获得13.1 mg proNGF蛋白。用透析复性的方法将目的蛋白重折叠, 复性率为18%, 在重折叠过程中前导肽发挥了一定的积极作用。用PC12细胞进行生物活性鉴定, 结果显示复性后的proNGF蛋白具有良好的生物活性。     

ProNGF在大肠杆菌中的表达、纯化和复性

将含有前导肽的人神经生长因子基因(proNGF)克隆在原核表达载体pET15b中, 转化大肠杆菌BL21(DE3)pLysS, 经IPTG诱导实现了目标融合蛋白的高效表达。SDS-PAGE分析表明表达蛋白占全菌总蛋白的20%左右, 表达蛋白主要以包涵体的形式存在。用6 mol/L的盐酸胍溶解包涵体后, 通过Ni2+-NTA柱纯化, 获得纯化的目标融合蛋白, 电泳谱带扫描分析表明蛋白纯度可达90%以上。Western blotting检测显示, 表达产物有较强的免疫学活性。经肠激酶作用后得到proNGF非融合蛋白, 分子量为27 kD, 100 mL表达菌液可获得13.1 mg proNGF蛋白。用透析复性的方法将目的蛋白重折叠, 复性率为18%, 在重折叠过程中前导肽发挥了一定的积极作用。用PC12细胞进行生物活性鉴定, 结果显示复性后的proNGF蛋白具有良好的生物活性。     

新型抗肿瘤转移多肽-三聚β肽(β３)在大肠杆菌中的表达及其抗粘附作用

自行设计了抗肿瘤转移多肽－三聚β肽（β3）,人工合成了β3的基因片段,构建了β3的表达质粒pET-His-β3,在大肠杆菌BL21(DE3)plysS中表达。在用IPTG诱导15h后可见明显的His-β3融合蛋白的表达,表达产物约占细胞总蛋白的4％,占细胞总不溶性蛋白的10％。每升pET-His-β3/BL21(DE3)plysS细菌培养液用金属螯合琼脂糖凝胶6B FF分离后可回收纯度为92.2%的β3产物约20mg。所表达出的β3肽对人肝癌细胞株SMMC-7721细胞及人肝癌高转移细胞株HCCLM6细胞与纤连蛋白（fibronectin, FN）粘附具有特异的抑制作用,呈现剂量效应相关关系和时间效应相关关系,抑制作用强于β肽（β1）、3倍浓度的β1（3×β1）和GRGDS。研究结果表明：pET-His-β3/BL21(DE3)plysS是β3适合的表达系统;表达的β3肽具有特异的抗肿瘤细胞粘附作用。     

棉铃虫颗粒体病毒增效蛋白基因2.1kb片段在大肠杆菌中的表达

以棉铃虫颗粒体病毒(Helicoverpa armigera granulosis virus,简称HaGV)基因组DNA为模板,设计引物PCR扩增病毒增效蛋白(Enhancin)基因,然后经SacI/PstI双酶切消化,得到5′端截短的约2.1kb增效蛋白基因片段,再与pQE30质粒连接,构建了重组表达载体pQE/EnC,转化大肠杆菌M15(pREP4),在IPTG诱导下表达出分子量约为78×10³ D的融合蛋白并命名为P78,纯化的P78包涵体显示了明显的增效活性,可提高AcMNPV对小菜蛾幼虫的感染率27.88%～32.92%。     

枯草杆菌DC-2纳豆激酶基因的克隆及其融合蛋白在E.coli中的表达

纳豆激酶是一种纤维蛋白溶解酶 ,有望开发成为新型的溶栓药物 .从中国豆豉中分离的具有较强纤溶活性的枯草杆菌DC 2中提取总DNA ,根据纳豆激酶 (NK)基因序列设计引物 ,用PCR法扩增NK基因 .序列分析表明 ,NK基因成熟肽编码区含有 82 5bp ,编码 2 75个氨基酸残基 ,与文献报道的序列分别有 93 4 %和 94 5 %同源性 .将NK基因插入载体pGEX 4T1构建表达质粒pGEX NK ,转化大肠杆菌JM10 9后 ,经 1mmol LIPTG诱导 4h ,发现大量NK融合蛋白表达 ,并形成包涵体 .SDS PAGE分析表明 ,NK融合蛋白作为包涵体的分子量为 5 3kD .凝胶自动扫描结果显示 ,NK融合蛋白约占菌体可溶性蛋白的 2 6 % .     

重组人骨形态发生蛋白2在CHO细胞中的表达、鉴定及活性分析

构建真核表达载体pCDNA3.1( )-hBMP-2,与质粒pSV2-dhfr共转染CHO-dhfr-细胞,以含有700μg/mLG418的IMDM进行选择性培养,筛选抗性克隆,并用MTX扩增,提高rhBMP-2的表达量。收集的rhBMP-2蛋白进行Westernblot检测,还原蛋白样品电泳产生一条大小约为18kD的特异性条带,非还原蛋白样品电泳产生一条大小约为30kD的特异性条带,提示表达的rhBMP-2是经过糖基化修饰的,且以同源二聚体形式分泌表达。单细胞分离培养得到14株rCHO(hBMP-2)单克隆细胞株,ELISA法检测rhBMP-2表达水平,最高可达7.83μg/24h/106cells。活性分析结果表明,表达的rhBMP-2具有很强的生物学活性。     

重组人骨形态发生蛋白－2成熟肽在大肠杆菌中的表达及其诱导成骨活性

报告了人骨形态发生蛋白－２（ＢＭＰ－２）成熟肽的基因克隆,及其在大肠杆菌中的表达。用ＡｆｌⅡ酶剪除ＢＭＰ－２ｃＤＮＡ中的信号肽及前肽部分的序列,将编码成熟肽的ｃＤＮＡ３′端０．３６ｋｂ基因片段克隆于大肠杆菌表达载体ｐＭＸ－１质粒的ＡＴＧ下游,受控于ＰＬＰＲ启动子。重组子以大肠杆菌ＤＨ５α为宿主细胞进行温度诱导表达。工程菌经４２℃６ｈ诱导后,在ＳＤＳ－ＰＡＧＥ上出现一条新蛋白带,分子量约为１２ｋＤ,约占菌体总蛋白的２０％。主要以包涵体形式存在的表达产物经初步纯化后,可获得纯度较高的重组人骨形态发生蛋白－２成熟肽（ｒｈＢＭＰ－２ｍ）。小鼠肌肉植入试验发现。ｒｈＢＭＰ－２ｍ植入后的不同时间,在局部出现间质细胞的聚集和增生、软骨细胞及软骨基质的生成和硬质骨的形成,证明ｒｈＢＭＰ－２ｍ具有明显的诱导新骨形成的作用。     

Systemically administered bone morphogenetic protein-6 restores bone in aged ovariectomized rats by increasing bone formation and suppressing bone resorption

Although recombinant human bone morphogenetic proteins (BMPs) are used locally for treating bone defects in humans, their systemic effect on bone augmentation has not been explored. We have previously demonstrated that demineralized bone (DB) from ovariectomized (OVX) rats cannot induce bone formation when implanted ectopically at the subcutaneous site. Here we showed in vitro that 17beta-estradiol (E2) specifically induced expression of Bmp6 mRNA in MC3T3-E1 preosteoblastic cells and that bone extracts from OVX rats lack BMPs. Next we demonstrated that 125I-BMP-6 administered systemically accumulated in the skeleton and also restored the osteoinductive capacity of ectopically implanted DB from OVX rats. BMP-6 applied systemically to aged OVX rats significantly increased bone volume and mechanical characteristics of both the trabecular and cortical bone, the osteoblast surface, serum osteocalcin and osteoprotegerin levels, and decreased the osteoclast surface, serum C-telopeptide, and interleukin-6. E2 was significantly less effective, and was not synergistic with BMP-6. Animals that discontinued BMP-6 therapy maintained bone mineral density gains for another 12 weeks. BMP-6 increased in vivo the bone expression of Acvr-1, Bmpr1b, Smad5, alkaline phosphatase, and collagen type I and decreased expression of Bmp3 and BMP antagonists, chordin and cerberus. These results show, for the first time, that systemically administered BMP-6 restores the bone inductive capacity, microarchitecture, and quality of the skeleton in osteoporotic rats.     

Heparin inhibits BMP-2 osteogenic bioactivity by binding to both BMP-2 and BMP receptor

Heparin demonstrates several kinds of biological activities by binding to various extracellular molecules and plays pivotal roles in bone metabolism. However, the role of heparin in the biological activity of bone morphogenetic protein (BMP) remains unclear. In the present study, we examined whether heparin has the effects on osteoblast differentiation induced by BMP-2 in vitro and also elucidated the precise mechanism by which heparin regulates bone metabolism induced by this molecule. Our results showed that heparin inhibited alkaline phosphatase (ALP) activity and mineralization in osteoblastic cells cultured with BMP-2. Heparin was found to suppress the mRNA expressions of osterix, Runx2, ALP and osteocalcin, as well as phosphorylation of Smad1/5/8 and p38 MAPK. Further, heparin bound to both BMP-2 and BMP receptor (BMPR). These results suggest that heparin suppresses BMP-2-BMPR binding, and inhibits BMP-2 osteogenic activity in vitro.     

Bone morphogenetic protein-2 converts the differentiation pathway of C2C12 myoblasts into the osteoblast lineage [published erratum appears in J Cell Biol 1995 Feb;128(4):following 713]

The implantation of bone morphogenetic protein (BMP) into muscular tissues induces ectopic bone formation at the site of implantation. To investigate the mechanism underlying this process, we examined whether recombinant bone morphogenetic protein-2 (BMP-2) converts the differentiation pathway of the clonal myoblastic cell line, C2C12, into that of osteoblast lineage. Incubating the cells with 300 ng/ml of BMP- 2 for 6 d almost completely inhibited the formation of the multinucleated myotubes expressing troponin T and myosin heavy chain, and induced the appearance of numerous alkaline phosphatase (ALP)- positive cells. BMP-2 dose dependently induced ALP activity, parathyroid hormone (PTH)-dependent 3',5'-cAMP production, and osteocalcin production at concentrations above 100 ng/ml. The concentration of BMP-2 required to induce these osteoblastic phenotypes was the same as that required to almost completely inhibit myotube formation. Incubating primary muscle cells with 300 ng/ml of BMP-2 for 6 d also inhibited myotube formation, whereas induced ALP activity and osteocalcin production. Incubation with 300 ng/ml of BMP-2 suppressed the expression of mRNA for muscle creatine kinase within 6 h, whereas it induced mRNA expression for ALP, PTH/PTH-related protein (PTHrP) receptors, and osteocalcin within 24-48 h. BMP-2 completely inhibited the expression of myogenin mRNA by day 3. By day 3, BMP-2 also inhibited the expression of MyoD mRNA, but it was transiently stimulated 12 h after exposure to BMP-2. Expression of Id-1 mRNA was greatly stimulated by BMP-2. When C2C12 cells pretreated with BMP-2 for 6 d were transferred to a colony assay system in the absence of BMP-2, more than 84% of the colonies generated became troponin T-positive and ALP activity disappeared. TGF-beta 1 also inhibited myotube formation in C2C12 cells, and suppressed the expression of myogenin and MyoD mRNAs without inducing that of Id-1 mRNA. However, no osteoblastic phenotype was induced by TGF-beta 1 in C2C12 cells. TGF-beta 1 potentiated the inhibitory effect of BMP-2 on myotube formation, whereas TGF-beta 1 reduced ALP activity and osteocalcin production induced by BMP-2 in C2C12 cells. These results indicate that BMP-2 specifically converts the differentiation pathway of C2C12 myoblasts into that of osteoblast lineage cells, but that the conversion is not heritable.     

Non-functional Fas ligand increases the formation of cartilage early in the endochondral bone induction by rhBMP-2

It has previously been shown that mice with a defect in Fas ligand-mediated apoptosis have an enhancement of ectopic bone formation. We investigated the expression of bone-related markers--alkaline phosphatase, collagen, bone sialoprotein, osteocalcin, osteopontin, and bone morphogenetic proteins (BMP) -2, -4, and -7; and cytokines interleukin-1alpha (IL-1), IL-1beta, and tumor necrosis factor-alpha (TNF-alpha) in ectopic new bone induced by recombinant human (rh) BMP-2 in mice without functional Fas-ligand (gld mice). At day 6 after rhBMP-2 implantation, gld mice formed more cartilage and mesenchyme compared with their wild type littermates. At later stages, gld mice did not differ from the control mice in the volume of newly formed tissue, expressing higher level of BMP genes and lower levels of genes involved in osteoblast maturation--bone sialoprotein and osteopontin. Differences in the levels of expression of IL-1alpha and TNF-alpha were observed only at day 12 after rhBMP-2 implantation. These results suggest that gld mice have an increased recruitment of cells of mesenchymal origin and an abnormal pattern of differentiation and maturation of the newly formed mesenchymal tissues.