猪生长激素cDNA在芽孢杆菌中的表达

利用随机克隆的枯草杆菌启动子-信号序列构建茅孢杆菌分泌载体pUS186。用限制酶将切除了信号序列的猪生长激素cDNA从质粒pLY3-PGH 604切下,亚克隆至pUS186,并在该cDNA的下游接上地衣杆菌α-淀粉酶基因的转录终止子,构建猪生长激素表达质粒pSGH 1864,将此质粒转化蛋白酶双缺陷的枯草杆菌DB104及短小茅孢杆菌289。SDS-聚丙烯酰胺凝胶电泳检出在发酵上清液中多出一条22kD的蛋白带,抗猪生长激素血清免疫印迹法证明这一蛋白带具有免疫活性,表明猪生长激素cDNA已在枯草杆菌及短小茅抱杆菌中表达。     

枯草杆菌α－淀粉酶基因的克隆和表达

以自构的质粒pBEl为载体,采用鸟枪法克隆枯草杆菌168突变株GRl0的α－淀粉酶基因,获得了在大肠杆菌中的表达。该基因片段位于7367bp的Bgl Ⅱ酶切片段上。利用质粒pUB110。将此片段亚克隆到枯草杆菌中,同样也获得了表达。本文还测定了该基因克隆子是否有GR10的抗葡萄糖阻遏效应。     

分泌载体pUS186的构建及地衣杆菌α—淀粉酶基因在枯草杆菌中的表…

利用枯草杆菌的分泌系统构建分泌型表达载体表达和分泌外源基因产物具有重要的商业价值。我们用鸟枪法克枯草杆菌染色体的启动子和信号肽序列。将克隆的序列连接到能在枯草杆菌中复制的质粒ｐＵＢ１８上,获得分泌型表达载体ｐＵＳ１８６。为了测试构建的载体ｐＵＳ１８６的功能,将地衣杆菌α－淀粉酶基因的缺失了启动子和信号肽序列的片段重组进该质粒,经过Ｂａｌ ３１酶切,Ｔ４ ＤＮＡ聚合酶补齐等处理,获得ｐＵＳＡ１８６Ｉ     

分泌载体pUS186的构建及地衣杆菌α－淀粉酶基因在枯草杆菌中的表达和分泌

利用枯草杆菌的分泌系统构建分泌型表达载体表达和分泌外源基因产物具有重要的商业价值。我们用鸟枪法克隆了枯草杆菌染色体的启动子和信号肽序列,将克隆的序列连接到能在枯草杆菌中复制的质粒ｐＵＢ１８上,获得分泌型表达载体ｐＵＳ１８６。为了测试构建的载体ｐＵＳ１８６的功能,将地衣杆菌α－淀粉酶基因的缺失了启动子和信号肽序列的片段重组进该质粒,经过Ｂａｌ３１酶切,Ｔ４ＤＮＡ聚合酶补齐等处理,获得ｐＵＳＡ１８６Ⅱ及ｐＵＳＡ１８６Ⅰ系列质粒,将这些重组质粒转化枯草杆菌ＱＢ１１３０（ａｍｙ－）后都能向胞外分泌淀粉酶,酶活测定结果表明,基因表达水平比用原有的启动子高１－２倍,蛋白质分泌率在８４－９６％之间。     

肿瘤坏死因子相关凋亡受体(TRAIL)cDNA的克隆及高效表达

为了得到人TRAILcDNA并克隆到pGEM-Tvector。利用PCR技术获得目的基因片段 ,通过原核表载体pBV220构建人TRAIL表达质粒。将所得表达质粒转化宿主菌DH5α ,培养至OD60 0 值到达 0 6时 42℃诱导表达 ,并通过SDS PAGE分析表达结果。经凝胶薄层扫描 ,对pBV TRAILD DH5α工程菌热诱导 5h ,TRAIL衍生物蛋白的表达量最高约占菌体可溶性总蛋白的 31 %。人TRAIL基因 ,通过pBV2 2 0原核表达载体可在大肠杆菌中获得高效表达。     

双功能枯草杆菌诱导型高效表达分泌载体的构建与鉴定

利用大肠杆菌质粒pSP72和枯草杆菌质粒pUB18共整合得到双功能克隆载体pSB。在pSB多克隆位点依次引入枯草杆菌果聚糖蔗糖酶基因启动子-信号肽序列sacBp.s.、地衣芽孢杆菌淀粉酶基因终止子序列α-amyT和短小芽孢杆菌增强子基因degQ,最终构建了双功能枯草杆菌诱导型高效表达分泌载体pSBPTQ。将VasostatinⅠ基因作为靶基因检测sacBp.s.、α-amyT和degQ在pSBPTQ进行外源基因表达时的功能,结果表明,在蔗糖诱导下,sacB启动子有效启动了Vasostatin I基因的表达和分泌,α-amy T提高了VasostatinⅠ基因的转录效率,而degQ明显增强了VasostatinⅠ基因的表达水平。VasostatinⅠ基因在蔗糖诱导下成功表达并分泌到枯草杆菌细胞外,蛋白质分泌效率达到90%左右。质粒稳定性试验结果表明,经过40个世代之后,质粒pSBPTQ在枯草杆菌DB1342中仍旧保持在83%以上。     

嗜铬粒蛋白N端片段基因在枯草杆菌中的表达及表达产物的活性分析

嗜铬粒蛋白(CGA)是存在于分泌细胞的由439个氨基酸组成的可溶性蛋白。近年的研究发现CGA的N端具有抗血管收缩、抗细菌和抗真菌的功能。为了寻找高效低毒的抗真菌片段,利用PCR技术扩增了编码人嗜铬粒蛋白N端1-76位氨基酸（CGA1-76）的DNA片段,并将之克隆进本实验构建的枯草杆菌诱导型表达载体pSBPTQ,获得含CGA1-76基因的重组质粒pSVTQ,转化蛋白酶三缺陷的枯草杆菌DB403。经蔗糖诱导后,CGA1-76片段在枯草杆菌DB403（pSVTQ）中获得表达,产物分泌到细胞外,分泌量约为5mg/L,占总分泌蛋白的133% 。测试了表达产物对几种丝状真菌和酵母的抑制作用,发现在4μmol/L的浓度下,枯草杆菌表达的重组CGA1-76对镰刀菌、链格孢霉及白假丝酵母有明显的抑制作用。     

部分扩增与双片段连接相结合制作长基因定点突变

重叠延伸PCR是基因定点突变的主要方法,但是以该方法制作长基因定点突变时,往往遇到难以获得第二轮PCR产物或容易引入新的非预期突变等问题。此时,可先以重叠延伸PCR扩增含突变位点的部分基因片段,再将其连入适当载体获得重组质粒。若该扩增片段两侧的酶切位点在质粒载体上不单一,则可采用双片段连接法构建完整质粒。以制作视网膜母细胞瘤基因S780E定点突变为例,直接以重叠延伸PCR扩增全长基因时未能得到理想的目标产物。故先扩增含点突变的F3片段,再将其与源自原始质粒的F2片段一起连入含F1片段的质粒载体而构建完整质粒。两个筛选出的重组质粒经序列检测完全符合目标突变序列特征,验证了该方案的可行性。该方法作为重叠延伸PCR的补充,可为许多长基因定点突变提供解决方案。     

一种构建马链球菌兽疫亚种血红素受体基因缺失突变株的方法

【目的】探讨一种构建马链球菌兽疫亚种基因缺失突变株的方法。【方法】PCR扩增目的基因,用pJR700温度敏感载体系统,构建目的基因载体;反向PCR扩增目的基因缺失载体片段,连接,产生目的基因缺失载体;电转化缺失重组质粒导入感受态细胞,先在37℃卡拉霉素(kan)培养基中连续培养,然后在30℃不含kan液体培养基中传代,挑取抗生素敏感菌落,PCR扩增检测抗生素敏感菌染色体上目的基因片段和链黑霉素抗性实验确认血红素受体基因缺失。【结果】获得不含抗生素基因的马链球菌兽疫亚种血红素受体基因缺失突变株。【结论】用pJR700温度敏感载体系统,构建马链球菌兽疫亚种基因缺失突变株是可行的。     

白念珠菌FLO8基因突变株构建及鉴定

目的构建白念珠菌FLO8基因突变株。方法将白念珠菌FLO8基因插入pCP20质粒载体ADH1启动子之后,通过定向诱变获得FLO8基因R209T、A311T、654Ter、G723R、T751D突变质粒载体,再通过同源重组技术将带有FLO8突变的基因片段整合至SN152flo8/flo8菌株的ADE2位点。结果通过测序鉴定FLO8基因突变质粒载体构建成功;通过PCR验证表明突变FLO8基因整合到SN152flo8/flo8菌株的ADE2位点。结论以pCP20质粒为载体,通过定向诱变、同源重组等技术,可以高效构建白念珠菌FLO8基因突变株。     

High-level expression and purification of human epidermal growth factor with SUMO fusion in Escherichia coli

Human epidermal growth factor (hEGF) can stimulate the division of various cell types and has potential clinical applications. However, the high expression of active hEGF in Escherichia coli has not been successful, as the protein contains three intra-molecular disulfide bonds that are difficult to form correctly in the bacterial intracellular environment. To solve this problem, we fused the hEGF gene with a small ubiquitin-related modifier gene (SUMO) by synthesizing an artificial SUMO-hEGF fusion gene that was highly expressed in Origami (DE3) strain. The optimal expression level of the soluble fusion protein, SUMO-hEGF, was up to 38.9% of the total cellular protein. The fusion protein was purified by Ni-NTA affinity chromatography and cleaved by a SUMO-specific protease to obtain the native hEGF, which was further purified by Ni-NTA affinity chromatography. The result of the reverse-phase HPLC showed that the purity of the recombinant cleaved hEGF was greater than 98%. The primary structure of the purified hEGF was confirmed by N-terminal amino acid sequencing and MALDI-TOF mass spectroscopy analysis. Using the method of methylthiazoletetrazolium, the mitogenic activity on Balb/c 3T3 cells of the purified hEGF was comparable to that of commercial hEGF.     

Human breast cancer cells synthesize and secrete an EGF-like immunoreactive factor in culture

A human breast cancer cell line, strain MCF-7, in culture synthesized and secreted a large amount of a polypeptide (designated as MCF-7 EGF) immunologically related to human epidermal growth factor (hEGF). The molecular weight of MCF-7 EGF estimated by gel filtration on Sephadex G-75 was similar to that of hEGF from human urine. On isoelectric focusing analysis, MCF-7 EGF gave a major peak at pH 4.6 and a minor peak at pH 5.0. In our enzyme immunoassay system, however, the dose-response curve of MCF-7 EGF did not show good parallelism with that of standard hEGF. From these results, it is suggested that MCF-7 cells synthesize and secrete a polypeptide immunologically related to hEGF into the culture medium.     

Secretion of human EGF and IgE in mammalian cells by recombinant DNA techniques; use of a IL-2 leader sequence

Expression plasmids were constructed containing chemically synthesized human epidermal growth factor (EGF) gene fused in a frame to a leader sequence of human interleukin-2 (IL-2) gene under the control of a viral promoter. COS7 cells transfected with the plasmids synthesized and secreted EGF. Transfection of mouse A9 cells or BALB/3T3 clone A31 cells with the plasmids permitted the isolation of cell lines secreting the product which showed EGF activity. In particular, A31 transformed cells secreting human EGF grew well even in a medium containing a minimal level of serum. Using similar vectors having IgE cDNA (C2-C4) in place of EGF gene, a human IgE Fc fragment was also produced and secreted in mouse cells. These results show that heterologous leader sequences are useful for the expression and secretion of proteins whose genes lack leader sequences.     

Characterization of a high-molecular-weight form of epidermal growth factor in an extract of human urine

We purified from a side fraction of the commercial preparation of urokinase from large volumes of human urine a high-molecular-weight (HMW) form of human epidermal growth factor (hEGF). Sequence analysis of the amino terminus of the intact molecule and of two tryptic fragments and carboxypeptidase Y analysis revealed the molecule to correspond to residues 828-1023 of the hEGF precursor predicted by the nucleotide sequence of human renal hEGF mRNA, with hEGF forming its carboxyl terminus. HMW hEGF bound poorly to concanavalin A-agarose, quite avidly to wheat germ lectin-agarose, and completely to phenyl boronate-agarose, suggesting that it was O-glycosylated. Sephacryl S-200 chromatography of freshly-voided urine revealed mostly hEGF, with smaller amounts of a much higher molecular weight hEGF, but little material that was the size of the HMW hEGF we characterized. The large fragment we characterized presumably is cleaved from the larger form by enzyme(s) present in urine during the collection, storage, and processing of urine. We have confirmed that hEGF is synthesized as a large precursor molecule, as predicted by the nucleotide sequence of hEGF mRNA.     

犬细小病毒VP2蛋白在真核细胞中的分泌表达及特性

摘要:【目的】利用真核细胞分泌表达犬细小病毒VP2蛋白和研究其特性。【方法】为构建犬细小病毒（Canine parvovirus, CPV）VP2基因的真核分泌型表达载体,首先通过酶切从含有人CD5信号肽序列的质粒中将CD5信号肽基因片段切出,将其连接到真核表达载体pcDNA3.1A的多克隆位点上,构建成pcDNA3.1-CD5sp质粒。然后再通过PCR方法从含有犬细小病毒VP2基因的质粒中扩增VP2基因,并将其插入到pcDNA3.1- CD5sp载体中CD5信号肽的下游,构建成VP2基因的真核分泌型表达载体pcDNA-CD5sp-VP2。经磷酸钙介导转染293T细胞,使其在真核细胞中进行分泌表达,并通过ELISA检测表达的VP2蛋白与犬转铁蛋白受体（TfR）结合的活性。【结果】序列分析结果表明,本实验构建的犬细小病毒VP2基因真核分泌型表达载体结构正确,将该表达载体转染的293T细胞,在培养基中通过Western-blot检测到有VP2重组蛋白的存在。经ELISA检测表明表达的重组VP2蛋白具有与犬转铁蛋白受体结合的活性。【结论】 利用人的CD5信号肽实现了犬细小病毒VP2蛋白在真核细胞中的分泌表达,表达的VP2蛋白具有与犬转铁蛋白受体结合的活性。     

Synthesis and expression of the gene for human epidermal growth factor in transgenic potato plants

Summary The gene for human epidermal growth factor (hEGF) was chemically synthesized and used for expression in transgenic potato. The hEGF coding sequence was modified by PCR to introduce ATG start codon and fused either to the 35S cauliflower mosaic virus promoter or to the patatin class I promoter. The highest hEGF peptide content, 120 pg/mg soluble protein, was found in potato tubers when the chimeric gene was expressed under the control of patatin promoter.     

Synthesis and secretion of an epidermal growth factor (EGF) by human fibroblast cells in culture

Human fibroblast (WS-1) cells in culture synthesized and secreted an epidermal growth factor which cross-reacted with human epidermal growth factor (hEGF) purified from human urine. hEGF secreted by the cells (designated as WS-1 EGF or fibroblast EGF) and hEGF isolated from urine (designated as urine EGF) were immunologically indistinguishable. The molecular weight of fibroblast EGF estimated by gel filtration was identical with that of hEGF from urine. On chromatofocusing chromatography, fibroblast EGF was eluted mainly at pH 4.26 as a sharp symmetric peak with a minor peak at pH 4.62, like urine EGF. These results suggested that EGF synthesized and secreted by human fibroblast cells is an identical molecule to that of hEGF in human urine.     

Synthesis and secretion of an hEGF-like immunoreactive factor by human gastric cancer cells (MKN-45)

A large amount of an immunoreactive factor was detected in the medium conditioned by human gastric cancer cells, strain MKN-45, by our enzyme immunoassay system for human epidermal growth factor (hEGF) based on hEGF isolated from urine. However, the dose-response curve of the immunoreactive factor (designated as MKN-45 EGF) was not parallel with the standard curve of hEGF. The molecular weight of MKN-45 EGF was slightly larger than that of hEGF and was estimated to be 7,000-8,000 by gel filtration on Sephadex G-50. On isoelectric focusing analysis, MKN-45 EGF gave a major peak at pH 5.0 and a minor one at pH 4.3. These results demonstrate that MKN-45 cells synthesize and secrete into the culture medium a polypeptide immunologically related to hEGF.