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重组人肝细胞生长因子改造后在酵母中二级结构活性的研究
来源:吕云福 | 作者:sjzxads | 发布时间:2012-8-26 访问人数: 232

重组人肝细胞生长因子改造后在酵母中二级结构活性的研究

 吕云福1  董永红2  宫晓光1 刘宁1  常顺伍1  邱庆安1  黄海1  王保春1

  

基金项目:海南省自然科学基金资助项目(琼科函[2006]291号)

 

(海南省人民医院普外科 ,山西省人民医院普外科)

[摘要] 目的 探讨人肝细胞生长因子(hHGF)改造后重组hHGF(rhHGF)蛋白在P.pastoris酵母中二级结构的活性。方法  根据酵母偏爱密码子和Kex2酶切识别位点,通过套叠PCR技术对hHGF cDNA进行改造并与pGEM-T Easy载体连接,蓝白斑筛选,XbaⅠ/SalⅠ双酶切鉴定后测序。构建穿梭载体pPICZα A-rhHGF,XbaⅠ/XhoⅠ酶切鉴定。将穿梭载体质粒用SacⅠ单酶切线性化,转化P.pastoris GS115细胞,表型鉴定,拷贝数确定。采用甲醇诱导表达,选择表达量最高的1株进行温度控制性优化表达。对表达产物进行Western blot分析和生物学活性测定。结果  XbaⅠ/SalⅠ双酶切获得2.1kb大小的片段,测序结果显示AAG序列插入到目的位置,其余序列正确。穿梭载体构建后,XbaⅠ/XhoⅠ双酶切鉴定显示为两条带,分别为580bp和1074bp,与预期一致。转化P.pastoris GS115细胞,PCR鉴定表型均呈mut+,Zeocin浓度筛选拷贝数均为1。选取其中6个转化菌甲醇诱导,表达rhHGF的浓度分别为14.5mg/L,14.6mg/L, 16.1mg/L,18.7mg/L, 19.3mg/L, 20.5mg/L。还原状态下,SDS-PAGE凝胶电泳显示为两条带。Western blot检测显示在约80kDa处有一条浓聚带,证实rhHGF表达产物免疫原性存在。结论 经过基因改造的rhHGF在P.pastoris 中获得表达,其表达产物的二级结构具备生物学活性,有增强HGF的作用,但表达量尚待进一步提高。

[关键词]  肝细胞生长因子;酵母/巴斯德毕赤;改造;活性;二级结构

Study on two-structure with biological activities of recombinant human hepatocyte growth factor in transformation.Department of General Surgery, Hainan Provincial people’s Hospital Lu Yunfu, Gong XiaoGuang,Department of General Surgery,shanxi Provincial people’s Hospital Dong Yonghong,et al.

 [Abstract] Objective  To probe into human hepatocyte growth factor(hHGF) cDNA by transformation, biological activities of two-structure. Methods The initial part of hHGF cDNA was modified according to optimal e­xpression codons and the connection part between α and β-chain was done according to protease expressed in Pichia pastoris itself. The rhHGF was ligated to pGEM-T Easy vector, which was sequenced after blue/white color screening and identification by XbaⅠ/SalⅠ cleavage. The interest gene cleavaged by XbaⅠ/SalⅠwas inserted into pPICZα A vector. The linearized pPICZα A-rhHGF plasmid DNA with SacⅠwas transformed into Pichia pastoris GS115 by  

electroporation. Phenotypes were identified by PCR, and copy number was screened on YPD plate containing different concentration of Zeocin. After induce e­xpression

with methanol, a production-improved method by lower temperature, was further applied to the most e­xpression strain. Immunity and biological activity of rhHGF was detected, respectively.  Results An about 2.1 kb DNA fragment was seen after XbaⅠ/SalⅠcleavage, and the sequenced result showed that AAG was inserted into the aimed site. The recombined pPICZα A-rhHGF was cleavaged into two bands,580 bp and 1074 bp as expected. Phenotypes identified by PCR were all mut+, and copy number was 1 screened on YPD plate containing different concentrations of Zeocin. Picking 6 colonies and inducing e­xpression with methanol, the concentration of rhHGF was 14.5mg/L, 14.6mg/L, 16.1mg/L, 18.7mg/L, 19.3mg/L,20.5mg/L,respectively. The result of SDS-PAGE gel showed to be two bonds under reduction state. A strong bond was seen at 80 kDa by Western blot under non-reduced state, which confirmed that rhHGF protein had a certain immunity. Conclusion hHGF cDNA was successfully modified by overlap PCR,and rhHGF was expressed in Pichia pastoris. Expressed rhHGF have biological activities of two-structure.The level of rhHGF e­xpression in Pichia pastoris is too low and to be improved.

[Key words] Hepatocyte growth factor; Pichia pastoris; transformation ; Activity;      two-structure

 

 

      肝细胞生长因子(HGF)是一种多功能细胞因子,具有促进肝细胞再生,抑制肝纤维化及肝细胞凋亡,溶解硬变肝脏纤维组织[1],减轻肝损害[2]、提高骨髓基质干细胞与纤维连接蛋白涂层的黏附行为[3]等功能。其活性结构为α链与β链通过二硫键连接而形成的异二聚体。我们拟就hHGF cDNA进行特异标识,将其在P.Pastoris酵母表达,利用后者分泌的蛋白酶将其切割,获得具有活性的二级结构的rhHGF蛋白,从而增强HGF的作用。

 

材料与方法

1.材料:E. coli. DH5α和E. coli. TOP10F’ 由周鹏博士实验室冻存。P.pastoris GS115(his4, Mut+)酵母细胞菌株购自Invitrogen公司。pRc/CMV-hHGF质粒,由山西医科大学程牛亮教授惠赠。毕赤酵母分泌表达型载体pPICZαA表达质粒购自Invitrogen 公司。小鼠抗人HGF单克隆抗体系US Biological 产品,山羊抗小鼠IgG-AP,NBT/BCIP染色试剂盒购自华美生物工程公司。

2.引物的合成:由上海生工生物工程公司合成四条引物,其核苷酸序列分别如下:P1:5’CTGTCGACAAAAGAGAGGCTGAAGCTCCAGCTTTGAAGATTAAGACTAAGAAGGTTAAT 3';  P2:5’ ACAATTGAAGCGAGTTGTAAATG 3';  P3:5’ CATTTACAACTCGCTT CAATTGT  3’;  P4:5’  

GTTCTAGATGACTGTGGTACCTTATATG  3’

3.rhHGF基因扩增:分别以P1和P3、P2和P4为引物,pRc/CMV -hHGF质粒为模板,PCR扩增出rhHGF的α链和β链;再以P1和P4为引物,rhHGF的α链和β链为模板,PCR扩增出rhHGF的全长链。

4.pGEM-T Easy-rhHGF质粒的构建:将回收的rhHGF  PCR产物和pGEM-T Easy Vector进行连接,转化入E.coli DH5α感受态细胞,蓝白斑筛选。提取质粒用Xba I和SalⅠ酶进行双酶切鉴定。阳性菌落进行基因测序。

5.pPICZαA-rhHGF载体的构建:目的基因序列正确后,将质粒用Xba I和SalⅠ双酶切下后与预酶切好的pPICZαA片段在16℃恒温环境中连接12h以上,然后转化E.coli TOP10F’感受态细胞,挑取菌落10个,在低盐LB+Zeocin培养基中培养,提取质粒,并XbaⅠ和XhoⅠ双酶切鉴定。

6.pPICZαA-rhHGF在GS115中的表达:穿梭载体质粒以SacⅠ酶切线性化后,用GenePulser转化入GS115感受态细胞,YPD+Zeocin 30℃培养箱,培养2d。取PCR鉴定后的阳性重组子接种于20mL BMGY液体培养基中,30℃振荡培养16-18h,至菌体OD600达到2-6时,离心弃上清,换25mL BMMY液体培养基。用甲醇诱导培养5d,取上清,SDS-PAGE电泳。取脱色条带清晰的凝胶,在MultiImage™ ⅡLight Cabinet下以BSA作为定量对照,直接读取蛋白表达值。

7.Western blot检测:用不加β-巯基乙醇的上样缓冲液重新行SDS-PAGE凝胶电泳,通过Fastblot快速电转仪进行蛋白质转移。先将转印膜进行封闭后,再分别与一抗、二抗反应,NBT/BCIP显迹。

 

 

结  果

1.rhHGF基因的PCR扩增:根据引物,分别在α链的5’端增加了SalⅠ酶切位点及α交配因子的信号肽酶切识别点,3’端增加了与β链套叠的碱基,因此PCR扩增后的α链长度应为1359bp;在β链的5’端增加了套叠的碱基,3’端增加了XbaⅠ酶切位点,因此PCR扩增后的β链长度为720bp。PCR扩增结果与理论推算值一致[4]。以改造后的rhHGF基因α链和β链为模板,P1和P4为引物PCR扩增,实际长度应当为2059bp。

2.pGEM-T Easy-rhHGF载体的构建:纯化回收产物与pGEM-T Easy连接,连接物转化入E.Coli DH5α中。经X-gal 蓝白斑筛选,结果见转化子大多呈白色菌落,说明转化效率高。挑取白色菌落LB+Amp培养基中振摇培养,并提取质粒,XbaⅠ和SalⅠ双酶切鉴定,切下的目的片段与理论值2059bp相符,见图1。选择一个条带最浓的阳性菌种送上海生工生物工程公司测序,结果与预期的完全一致。

3. pPICZαA-rhHGF载体的构建:对转化入E.Coli TOP10F’ 的连接物经Zeocin压力筛选,菌落PCR鉴定后,取阳性菌落于低盐LB+Zeocin中培养,用 

XbaⅠ和XhoⅠ双酶切提取的质粒,结果见质粒被切成两条带,一条在600bp左右,另外一条在1100bp左右(见图2),而不再是单一的一条带。选取质粒酶切条带较浓的一个阳性带送测序,结果显示连接方向及插入位点完全正常。

4. pPICZαA-rhHGF在GS115中的表达:将SacⅠ酶切完全线性化的pPICZαA-rhHGF电转化法转入P.pastoris GS115细胞中。挑取10个转化子,在YPD+ Zeocin液体培养基中培养2d。分别提取酵母基因组DNA,用AOX1上、下游引物进行PCR扩增确定mut类型。10个中有9个转化成功,均为mut+:显示AOX1的2.2kb条带和单交换的2.2kb+580bp带。

   (略)

选择PCR亮度较高的6个电转化菌株,在BMGY中培养增殖,然后再移入BMMY中诱导表达。SDS-PAGE凝胶电泳(上样缓冲液中不加β-巯基乙醇),用BSA做定量对照,在 Alpha-Innotech DE500凝胶成像系统下直接读取表达量浓度值。表达量分别为14.5,14.6,16.1,18.7,19.3,20.5mg/L。以含β-巯基乙醇的上样缓冲液上样,SDS-PAGE凝胶电泳。结果如图3。Western blot检测结果如图4。

(略)

 

 

讨  论

活性hHGF是由α链与β链通过二硫键连接而成的异二聚体结构。为使表达产物具有活性二级结构,本试验在设计引物时,在α链与β链连接处利用双酶切法,借助于套叠PCR[5,6]手段对HGF基因表达产物进行重点改造。

由于SalⅠ与XhoⅠ属于同尾酶,故由pGEM-T Easy-rhHGF双酶切切下的片段,连到经XbaⅠ和XhoⅠ双酶切的pPICZαA载体上后,无论是XbaⅠ/SalⅠ还是XbaⅠ/XhoⅠ均无法再将原基因完整切下。但hHGF基因序列中本身即含有两个XhoⅠ酶切位点,因此,用XbaⅠ和XhoⅠ双酶切重组质粒pPICZαA-rhHGF会切出两条带。分析pPICZa A 载体多克隆位点区域可以发现,存在两处XhoⅠ位点,一个位于多克隆位点上方的α因子信号肽区域,另一个位于多克隆位点区域内的偏下游区。在设计时HGF的5’端引物带有α因子信号肽的酶切位点,如果目的基因插入到上游的XhoⅠ,则插入正确,表达的蛋白为目标蛋白,而如果插入下游的XhoⅠ处,则不但插入位置错误,而且表达的蛋白也会因为基因移码而变得面目全非。为了使之能正确插入,在双酶切pPICZa A 载体时,加入的酶量一定要足够,酶切时间要保证。其后对载有目的基因的质粒经过双酶切或PCR鉴定,只能属初步鉴定;为防止可能出现的错误连接或缺失,确保插入序列和预期完全一致,最好对重组质粒进行测序鉴定[7]。本试验构建的重组质粒pPICZαA-rhHGF经测序,结果显示插入序列与预期完全一致。

此外,在所构建的分泌型表达载体中,pPICZα A-rhHGF的前面含有α因子前导肽,所表达的融合蛋白rhHGF在α因子的作用下被成功地分泌到胞外[8]。在信号肽的后面,有能够被内切酶Kex2识别的位点和Stel3蛋白酶识别的Glu-Ala位点[9,10],在周间质α因子被有效切割。α链和β链连接处经基因改造增加了Kex2识别位点,在蛋白分泌后也被Kex2切断,起到了HGF在机体内被HGFA切割加工而产生活性结构的作用。因此,分泌到培养基介质中的蛋白应该是与天然HGF前体在机体内被加工后的蛋白结构相一致的表达产物。依据表达产物rhHGF的分子量和分泌到胞外的结果,进一步表明重组蛋白的前导信号肽被正确切割。

SDS-PAGE凝胶电泳时变性剂和表面活性剂是用来变性蛋白、暴露和溶解其疏水区域的。为了让大多数蛋白完全展开,有必要还原二硫键。本实验采用β-ME,使其解开二硫键,加之在上游基因改造时于α链和β链的连接处引入了Kex2酶切位点,因此在SDS-PAGE图上显示为两条带。而在供Western blot用的SDS-PAGE上样缓冲液中未加入β-巯基乙醇,α链和β链仍可依靠二硫键来维系,故此仅呈一条带。

虽然表达的rhHGF在还原状态下呈两条带,提示表达产物的二级结构具备生物学活性,有增强HGF的作用[11],但表达产量尚难以达到工业化生产的标准,尚需进一步研究。

 

参考文献

[1]  Ueki T,Okamoto E,Takeuchi M,et al.Persectives on postgenome medicine:Gene therapy for liver cirrhosis. Nippon Rinsho, 2001,59:152-156.

[2]  Xue F,Takahara T,Yata Y,et al.Attenuated acute liver injury in mice by naked hepatocyte growth factor gene transfer into skeletal muscle with electroporation.Gut,2002,50: 558-562.

[3]  黄盛东,刘晓红,白辰光,等. 肝细胞生长因子对骨髓基质干细胞与纤维连接蛋白涂层黏附行为的影响.中华实验外科杂志,2006,23:218-220.

[4]  Cruzado JM,Lloberas N,Torras J,et al.Regression of advanced diabetic nephropa hepatocyte rowfaccor gene theapy in rats.Diabetes,2004,53(4):1119-1127.

[5]  Pogulis RJ, Vallejo AN, Pease LR. In vitro recombination and mutagenesis by overlap extension PCR. Trower MK ed.Methods in molecular biology.Totowa NJ: Humana press Inc, 1995. 167-176.

[6] 周鹏,郭安平,黎小瑛.利用套叠PCR技术改造酵母表达载体pAO815的研究.生命科学研究, 2002,6:310-314.

[7] Dikmen E,Kara M,Kisa U,et al.Human hepatocyte growth factor levels in patients undergoing thoracic operations.Eur Respr J,2006,27(1):73-76.

[8]  Francone TD,Landmann RA,Chen CT,et al.Novel xenograft model expressing human hepatocyte growth factor shows ligad-dependent growth of c-Met-expressing tumors.Mol Cancer Ther.2007,6(4):1460-1466.

[9]  Kujan J, Herskowitz I. Structure of a yeast pheromone gene(MF-alpha):a putative alpha-factor precursor contains four tandem copies of mature alpha-factor. Cell,1982,30:933-943.

[10] Martinez-Ruiz A,Martinez del Pozo A,Lacadena J,et al.Secretion of recombinant pro- and mature fungal alpha-sarcin ribotoxin by the methylotrophic yeast Pichia pastois: the Lys-Arg motify is required for maturation.Protein Expr Purif,1998,12:315-322.

[11] Zuu J,Chen Y..Effect of human hepatocyte growth factor gene-modified bone marrow mesenchymal stem cells on immunological rejection after allograft liver transplantation in rats .Diabetes,Zhongguo Xiu, Fu Chong ,Jian Wai ,Ke Za Zhi,2011,25(7):871-876.