Received
2001-06-22Accepted 2001-08-18
This work was
supported by the National Natural
Science Foundation of China (Grant No.39800275)
*Corresponding
author. Tel: +86-10-63291921; Fax: +86-10-63051130; E-mail: anwei@cpums.edu.cn
生理学报, Feb. 2002,
54 (1): 23～2723
Acta
Physiologica Sinica
研究论文
重组人肝刺激物在原核细胞中的表达与纯化
杜海军, 孙红柳, 陈莉, 安威*
首都医科大学细胞生物系, 北京 100054
摘要: 利用基因重组技术, 构建成人肝刺激因子(hHSS)和谷胱甘肽转移酶(GST)融合表达载体,
转化大肠杆菌BL-21 (DE3), 以His·Tag亲和层析纯化表达产物, Factor Xa切割分离hHSS单体, 并检测其生物学活性。 结果显示, 在pET-42a表达体系中hHSS以可溶性蛋白和包涵体两种形式存在,
GST-hHSS表达量占菌体可溶性蛋白的30%; Factor Xa切割GST与hHSS之间肽腱, 得到33 和15 kD两条蛋白带, 经Western杂交证实33
kD条带为GST, 而15 kD条带的分子量与hHSS基因序列推测蛋白结果相符。经His·Tag再次纯化可获得hHSS单体, 初步证实重组hHSS具有促进肝癌细胞增殖活性。
关键词: 人肝刺激因子, 融合蛋白, 原核表达
学科分类号: Q485; R333.4
Prokaryotic expression and purification of human hepatic stimulator Substance
DU Hai-Jun,
SUN Hong-Liu, CHEN Li, AN Wei*
Department of
Cell Biology, Capital University of Medical Sciences, Beijing 100054
Abstract: To explore the possibility of
prokaryotic expression of human hepatic stimulator substance (hHSS), hHSS gene
was inserted in the downstream of glutathion S-transferase (GST) in a pET-42a
expression vector and recombinant GST-hHSS fusion protein was expressed under
IPTG induction in BL-21(DE3) cells. The recombinant HSS was purified with His·Tag
affinity chromatography, and its bioactivity was analyzed. The results showed
that GST-hHSS fusion protein was expressed both as a soluble or a inclusive body in bacterial cytosol.
The soluble GST-hHSS expression reached up to 30% of the whole soluble protein
of bacteria as determined by densitometry. The cleavage of GST-hHSS fusion
protein with Factor Xa produced two fragments of the protein, which sized 33 and 15 kD, respectively. The molecular weight of recombinant
HSS protein was identical to theoretical deduction based on the DNA sequences.
The protein homology of 15 kD hHSS could be efficiently eluted out after Factor
Xa cleavage. It is further indicated that the recombinant hHSS is able to
proliferate hepatoma cells of BEL-7402 in the preliminary experiments.
Key
words: human hepatic stimulator
substance; prokaryotic expression
; fusion protein
肝刺激因子(hepatic
stimulator substance, HSS)是广泛存在于初断乳动物肝脏或再生肝脏中的一种促肝细胞增殖因子[1～2]。有关HSS的研究虽取得了一定进展[3～7], 但由于它在肝细胞中含量极低, 提取和纯化十分困难, 至今仍未获纯品, 故迄今许多实验仍采用部分纯化的HSS。这不仅严重制约了HSS作用机制的深入研究,
也影响了其在临床的实际应用。鉴此, 本实验构建了hHSS原核表达载体, 体外表达重组hHSS, 为深入研究HSS的调控机制和临床应用提供了物质基础。
1材料和方法
1.1 质粒的转化与筛选分别以SalⅠ/EcoRⅠ消化表达载体pET-42a(+)(Novagen)和含hHSS基因的pUC18-hHSS (本室构建)。通过Glassmilk (Bio 101)回收相应片段, 构建pET-42a-hHSS表达质粒,
转化BL-21感受态细菌。以Kanamycin 筛选阳性细菌克隆, 再以PCR方法鉴定转化细菌是否含有hHSS基因, 条件为: 94℃ 变性5 min后,
94℃, 50 s; 65℃, 40 s; 72℃, 1 min 40 s; 反应30次循环; 最后72℃延长8 min。扩增产物经琼脂糖电泳确证含有hHSS重组基因的阳性克隆。
1.2 GST-hHSS融合蛋白的诱导与表达分别取5个含pET-42a-hHSS转化克隆菌, 接种LB培养液, 以IPTG (1 mmol/L)诱导hHSS基因表达。4
h后,以10000 g 4℃离心3 min收取细菌。加入变性液[5 mmol/L imidazole, 0.5 mol/L NaCl, 20 mmol/L
Tris-HCl (pH 7.9), 6 mol/L Urea]悬浮沉淀, 以超声细胞破碎仪裂解细菌。10000 g 4℃离心3 min, 取上清, 以BCA (bicinchoninic
acid)方法测定蛋白浓度。通过12% SDS-聚丙烯酰胺凝胶电泳(polyacrylamide gel electrophoresis, PAGE)观察不同克隆菌株中GST-hHSS表达。
1.3 GST-hHSS存在部位及表达形式的确定取高表达克隆菌株, 按方法1.2中步骤诱导GST-hHSS融合蛋白的表达, 离心制备细菌沉淀, 提取可溶性蛋白和包涵体蛋白。取诱导前细菌总蛋白、
诱导后培养液上清、 诱导后可溶性蛋白和包涵体蛋白各50 μg, 经12% SDS-PAGE分离, 用考马斯亮蓝(R-250)染色, 观察融合蛋白表达部位与形式。另取上述平行样品蛋白各5
μg, 按照狭缝杂交法将蛋白印渍于尼龙膜。该膜经5%脱脂奶粉过夜封闭(4℃), 次日与抗多聚组氨酸(His·Tag)抗体(Novagen)杂交1 h, 继而再与辣根过氧化物酶标记羊抗鼠IgG(Sigma)孵育1
h。之后, 将膜充分漂洗, 迅速加入发光试剂(Santa Cruz) 进行化学发光(enhanced chemiluminescence, ECL), 检测杂交信号强度。
1.4 GST-hHSS融合蛋白的纯化与含量的测定 将方法1.3中提取的可溶性蛋白和包涵体蛋白, 以0.45 μm滤膜过滤, 按His·Tag亲合层析方法分离融合蛋白。融合蛋白的His·Tag与亲合层吸柱中Ni2+发生耦联而被吸附,
先以低浓度咪唑液(60 mmol/L)洗脱去除杂蛋白, 再以高浓度咪唑(1 mol/L)洗脱亲合的目的蛋白, 即可获得纯化的GST-hHSS融合蛋白。取诱导前细菌总蛋白(30
μg)、 未纯化的细菌可溶性蛋白及包涵体蛋白(各30 μg)、 纯化的可溶性融合蛋白及包涵体融合蛋白(各3 μg), 经12% SDS-PAGE电泳分离, 用考马斯亮蓝(R-250)染色。以GS-700密度扫描蛋白条带密度,
计算GST-hHSS融合蛋白占BL-21菌体蛋白的百分含量。
1.5 GST-hHSS融合蛋白酶切取纯化GST-hHSS可溶性蛋白, 以Factor Xa 酶切缓冲液4℃过夜透析。次日, 取透析后纯化的可溶性GST-hHSS融合蛋白60
μg, 分成6管, 其中1管为阳性对照, 不加酶处理, 其余5管依次加入0.2、 0.4、 0.6、 0.8、 1 U 的Factor Xa; 21℃酶切24
h, 取3 μg酶切融合蛋白, 以12% SDS-PAGE凝胶电泳, 比较不同浓度Factor Xa酶切效果。酶切后的GST-hHSS以上述方法再次纯化, 进行电泳分离。
1.6 重组hHSS生物活性的研究采用MTT法[8]观察重组hHSS促细胞增殖状况。人肝癌细胞系BEL-7402以含10%胎牛血清(HyClone)的DMEM(GIBCO/BRL)培养基培养,
按1×104/ml细胞密度接种于96孔培养板, 于37℃、 5% CO2培养箱中培养。待细胞完全贴壁后, 换用无血清DMEM培养基(内含10 μg/ml胰岛素)培养12
h。将终浓度为0、 160、 240、 320、 400 ng/ml的重组hHSS加入培养基, 孵育24 h后用六孔重复测定其促细胞增殖活性。另设空白对照, 只加等体积培养基,
但不含重组hHSS。每孔加入20 μl MTT溶液(5 mg/ml, 北京华美生物工程公司)继续孵育4 h。以酶标仪(Microplate Reader
550, Bio-Rad)测定490 nm波长处各孔的吸光值。
2结果
2.1 含hHSS基因转化细胞的筛选
从pUC18-hHSS酶切得到hHSS cDNA, 应用Glassmilk回收纯化后, 正向插入pET-42a的EcoRⅠ和SalⅠ位点。通过卡那霉素筛选,
PCR扩增确证hHSS基因正确连接于pET-42a (图1), 并成功转化到BL-21细菌中。2.2 GST-hHSS融合蛋白表达形式及含量的测定
不同克隆菌株以IPTG诱导GST-hHSS融合蛋白的表达。诱导后融合蛋白表达量明显高于诱导前水平,
不同克隆间融合蛋白的表达量也不尽一致(图未示), 说明相同条件下各转化细菌间融合蛋白的表达效率差异较大。 挑选 hHSS 高表达克隆菌的诱导前细菌总蛋白、诱导后培养液上清、诱导后细菌胞浆可溶性蛋白和包涵体蛋白进行SDS-PAGE,
以抗His·
图1. pET42/GST-hHSS融合蛋白表达载体的构建
Fig.
1.Construction of GST-hHSS fusion gene in the expression vector pET42a(+).
Tag抗体进行蛋白狭缝杂交(图2A)。结果显示, GST-hHSS不仅存在于细菌胞浆的可溶性蛋白中, 也存在于包涵体中。经GS-700密度扫描仪分析(图2B),
GST-hHSS占可溶性蛋白中含量的30%。
图2.融合蛋白的印迹杂交鉴定和电泳分析
Fig.
2.Analysis of GST-hHSS fusion protein. Panel A represents slot blot of the
fusion protein in soluble fraction and inclusion body and panel B stands for
12% SDS-PAGE. Lane 1 is the total
cytosol-protein of bacteria before IPTG induction. Lane 2 is the LB culture
medium of bacteria after IPTG induction. Lanes 3 and 4 represent soluble
protein and inclusion body of bacteria after IPTG induction.
2.3 GST-hHSS融合蛋白的酶切纯化及活性分析
用Factor Xa 缓冲液过夜透析融合蛋白后以不同浓度酶切分析, 结果如图3所示。在33 和15 kD 处有两条明显的染色条带, 分别与GST蛋白和hHSS蛋白的分子量一致。如未经透析处理,
则Factor Xa的切割效率显著降低(图未示)。为进一步验证GST-hHSS融合蛋白, 以Western杂交方法对其进行分析。图4A显示, 酶切前的GST-hHSS融合蛋白及酶切后GST片段均与抗His·Tag抗体杂交,
说明融合蛋白GST-hHSS和GST片段中含多聚组氨酸, 这与pET42-a载体中GST分子量一致。Factor Xa酶切处理融合蛋白, 经His·Tag再次层析分离,
可获得纯化的hHSS单体分子(图4B), 其分子量约为15 kD, 与HSS基因序列推测的蛋白分子量相符, 进一步确证GST-hHSS融合蛋白表达、 纯化及酶切分析策略准确无误。
图3.融合蛋白经不同浓度Factor Xa切割
Fig. 3. Cleavage of the fusion protein with Factor Xa. GST-hHSS fusion
protein, dialyzed at 4℃, is digested by Factor Xa for 24 h and separated with
12%SDS-PAGE. Lane 1 represents
the protein molecule weight
marker. Lanes 2 to 6 are the
results of cleavage with 0.2 to 1 U of Factor Xa.
2.4 重组hHSS促细胞增殖的活性
我们曾报道, 生化提取的HSS可刺激体外培养的肝细胞增殖[9]。本实验将重组hHSS加入BEL-7402肝癌细胞系, 观察其作用效果, 以此作为鉴定重组hHSS生物活性的指标之一。如图5所示,
在160～400 ng/ml的刺激下, BEL-7402细胞的增殖加快, 并呈现出剂量-效应反应。在400 ng/ml作用24 h后, hHSS刺激组细胞OD490值较对照组升高34%,
统计学分析表明两组数值具有显著性差异(P<0.01), 提示重组HSS表现出明显促细胞增殖活性。
图4.GST-hHSS检测与纯化
Fig.
4.Identification and purification of GST-hHSS. In panel A, the purified fusion
protein of GST-hHSS either with Factor Xa cleavage (lane 1) or without cleavage
(lane 2) is separated with 12% SDS-PAGE and immunoblotted by anti-His·Tag
antibody. Lanes 1 to 3 in panel B represent uncut GST-hHSS, cut GST-hHSS with
Factor Xa and purified hHSS.
图5.重组hHSS刺激BEL-7402细胞增殖
Fig. 5.The
proliferation of BEL-7402 cells 24 h after stimulation by recombinant hHSS. The bioactivity
of recombinant hHSS is assayed by using MTT method. *P<0.01 vs control.
3讨论
利用DNA重组技术生产用传统方法难以获得的生物活性分子, 是近年分子医学和医药工程的热点研究课题。我们曾进行hHSS基因原核表达的研究, 并获得rhHSS,
但多以不溶性包涵体形式存在。从包涵体中进行蛋白复性, 只有少部分蛋白得以复性, 具有活性功能。复性过程是否会影响rhHSS的构象还无法确定。以融合蛋白的形式表达目的蛋白,
可增加外源蛋白可溶性和稳定性。通过His·Tag亲和层析方法纯化目的蛋白, 有利于提高重组蛋白的产量和纯度。以GST融合蛋白技术表达外源基因已有研究[10～13],
但有关GST-hHSS表达、 纯化及酶切分离方面尚未见报道。本研究发现, 人肝刺激因子不仅以包涵体形式, 还以可溶性蛋白形式存在于细菌中。实验表明,
GST-hHSS占可溶性蛋白含量的30%, 纯化后GST-hHSS的浓度可达2.6 mg/ml。提取和纯化可溶性GST-hHSS融合蛋白, 避免了变性-复性等复杂步骤,
减少了对蛋白构象的影响, 增加了蛋白的稳定性[14]。 Factor Xa切割GST-hHSS融合蛋白可获得hHSS单体。Factor Xa活性受离子浓度、
pH值、 温度、 时间、 变性剂、 酶用量及蛋白质本身属性等多种因素影响。本实验证实, 经过酶切缓冲液透析这一简单步骤处理, 可明显提高Factor Xa切割GST-hHSS的效果,
对这一改进还未见到有关报道。
本实验首次报道重组hHSS融合蛋白的表达和纯化,
并通过对rhHSS活性初步检测, 证明重组hHSS具有促肝癌细胞BEL-7402生长的作用, 为进一步研究基因重组HSS的功能和作用机制提供了物质基础。
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