CN114380897B - 一种结核分枝杆菌包涵体蛋白复性的方法及其专用复性缓冲液 - Google Patents
一种结核分枝杆菌包涵体蛋白复性的方法及其专用复性缓冲液 Download PDFInfo
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- CN114380897B CN114380897B CN202210035625.6A CN202210035625A CN114380897B CN 114380897 B CN114380897 B CN 114380897B CN 202210035625 A CN202210035625 A CN 202210035625A CN 114380897 B CN114380897 B CN 114380897B
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
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- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/35—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
Landscapes
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
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- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
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- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Gastroenterology & Hepatology (AREA)
- Microbiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
Abstract
本发明公开了一种结核分枝杆菌包涵体蛋白复性的方法及其专用复性缓冲液。实验证明,采用本发明提供的复性缓冲液可以复性结核分枝杆菌蛋白W544蛋白,复性得到的W544蛋白能有效刺激结核分枝杆菌感染小鼠的脾淋巴细胞产生免疫反应,同时不刺激健康小鼠的脾淋巴细胞产生免疫反应。由此可见,复性的W544蛋白具有很好的免疫原性。本发明提供的复性缓冲液及依据其建立的结核分枝杆菌包涵体蛋白复性方法具有重要的应用价值。
Description
技术领域
本发明属于蛋白纯化技术领域,具体涉及一种结核分枝杆菌包涵体蛋白复性的方法及其专用复性缓冲液。
背景技术
加强对结核病的诊、防、治研究对结核病的疫情控制有极其重要的意义。结核分枝杆菌蛋白在结核病的基础研究和开发应用中应用广泛,由于天然来源的结核分枝杆菌蛋白受到极大限制,采用基因工程技术几乎成为获取结核分枝杆菌蛋白唯一的有效手段。
用大肠杆菌系统表达蛋白具有易于操作、遗传背景清楚和表达水平高等特点,但使用大肠杆菌表达结核分枝杆菌蛋白时,表达产物多为不溶解、无活性的固体颗粒,即包涵体,需要对包涵体进行复性才能获得有活性的可溶蛋白。包涵体复性是一个复杂的过程,其成功与否很大程度上取决于蛋白的各种理化性质,尤其与蛋白序列中半胱氨酸的含量有关,半胱氨酸含量越多,复性越困难,多数含半胱氨酸的包涵体蛋白无法采用透析法及稀释法等常规方法进行复性,并因此而影响后续相关研究的进展及产品的开发。因此,建立有效的结核分枝杆菌包涵体蛋白复性方法极其重要。
发明内容
本发明的目的是如何对结核分枝杆菌包涵体蛋白进行复性。
本发明首先保护一种结核分枝杆菌包涵体蛋白复性的方法,可包括如下步骤:
(1)将结核分枝杆菌包涵体蛋白和复性缓冲液混合,静置,得到复性产物;
所述复性缓冲液的溶质及其浓度为左旋精氨酸0.8-1.2M(如0.8-1.0M、1.0-1.2M、0.8M、1.0M或1.2M)、氯化钠0.1-0.3M(如0.1-0.2M、0.2-0.3M、0.1M、0.2M或0.3M)、三羟甲基氨基甲烷0.03-0.07M(如0.03-0.05M、0.05-0.07M、0.03M、0.05M或0.07M)、β-环状糊精1.3-1.7%(如1.3-1.5%、1.5-1.7%、1.3%、1.5%或1.7%)、Tween200.05-0.15%(如0.05-0.10%、0.10-0.15%、0.05%、0.10%或0.15%),溶剂为水,pH7.5-8.5(如pH7.5-8.0、pH8.0-8.5、pH7.5、pH8.0或pH8.5);
(2)完成步骤(1)后,将复性产物进行超滤,得到的上清液即为结核分枝杆菌蛋白溶液。
上述方法中,所述步骤(1)具体可包括(1-1)和(1-2):
(1-1)将1体积份结核分枝杆菌包涵体蛋白溶液和(0.5-1.5)体积份(如(0.5-1.0)体积份、(1.0-1.5)体积份、0.5体积份、1.0体积份或1.5体积份)所述复性缓冲液混合,3-5℃(如3-4℃、4-5℃、3℃、4℃或5℃)静置0.5h-1.5h(如0.5h-1.0h、1.0h-1.5h、0.5h、1.0h或1.5h);
(1-2)向完成步骤(1-1)的体系中加入(2.5-3.5)体积份(如(2.5-3.0)体积份、(3.0-3.5)体积份、2.5体积份、3.0体积份或3.5体积份)所述复性缓冲液,混合,3-5℃(如3-4℃、4-5℃、3℃、4℃或5℃)静置0.5h-1.5h(如0.5h-1.0h、1.0h-1.5h、0.5h、1.0h或1.5h),得到复性产物。
上述方法中,所述步骤(2)具体可包括(2-1)和(2-2):
(2-1)将所述复性产物装入超滤离心管,离心超滤;
(2-2)将完成步骤(2-1)的体系继续离心超滤2次以上(如2次或3次),获得上清液;每次超滤的方法为:加入所述复性缓冲液,离心超滤。
上述任一所述复性缓冲液的溶质及其浓度可为左旋精氨酸1M、氯化钠0.2M、三羟甲基氨基甲烷0.05M、β-环状糊精1.5%、Tween200.1%,溶剂为水,pH8.0。
上述任一所述结核分枝杆菌包涵体蛋白的制备方法可如下:
(a1)向表达载体的多克隆位点插入编码结核分枝杆菌蛋白的基因,得到重组质粒;
(a2)将所述重组质粒导入大肠杆菌,得到重组菌;
(a3)发酵培养重组菌,经破碎、收集沉淀、洗涤、离心后获得结核分枝杆菌包涵体蛋白。
上述任一所述结核分枝杆菌蛋白可为W544蛋白;W544蛋白的氨基酸序列如SEQ IDNO:1所示。
上述任一所述编码W544蛋白的基因可如SEQ ID NO:2自5’末端起第7至2010位所示。
本发明还保护一种复性缓冲液,其溶质及其浓度为左旋精氨酸0.8-1.2M(如0.8-1.0M、1.0-1.2M、0.8M、1.0M或1.2M)、氯化钠0.1-0.3M(如0.1-0.2M、0.2-0.3M、0.1M、0.2M或0.3M)、三羟甲基氨基甲烷0.03-0.07M(如0.03-0.05M、0.05-0.07M、0.03M、0.05M或0.07M)、β-环状糊精1.3-1.7%(如1.3-1.5%、1.5-1.7%、1.3%、1.5%或1.7%)、Tween200.05-0.15%(如0.05-0.10%、0.10-0.15%、0.05%、0.10%或0.15%),溶剂为水,pH7.5-8.5(如pH7.5-8.0、pH8.0-8.5、pH7.5、pH8.0或pH8.5);
所述复性缓冲液用于结核分枝杆菌包涵体蛋白的复性。
上述任一所述复性缓冲液的溶质及其浓度具体可为左旋精氨酸1M、氯化钠0.2M、三羟甲基氨基甲烷0.05M、β-环状糊精1.5%、Tween200.1%,溶剂具体可为水,pH8.0。
上述任一所述复性缓冲液在复性结核分枝杆菌包涵体蛋白中的应用也属于本发明的保护范围。
实验证明,采用本发明提供的复性缓冲液可以复性W544蛋白,复性得到的W544蛋白能有效刺激结核分枝杆菌感染小鼠的脾淋巴细胞产生免疫反应,同时不刺激健康小鼠的脾淋巴细胞产生免疫反应。由此可见,复性的W544蛋白具有很好的免疫原性。本发明提供的复性缓冲液及依据其建立的结核分枝杆菌包涵体蛋白复性方法具有重要的应用价值。
附图说明
图1为实施例中步骤三最终获得的上清液、诱导前样品和诱导后样品的SDS-PAGE结果。
图2为实施例中步骤四酶联免疫斑点法(ELISPOT)检测W544蛋白的活性。
图3为对比例1中最终获得的上清液、诱导前样品、诱导后样品和复性产物超滤时的沉淀物的SDS-PAGE结果。
具体实施方式
下面结合具体实施方式对本发明进行进一步的详细描述,给出的实施例仅为了阐明本发明,而不是为了限制本发明的范围。以下提供的实施例可作为本技术领域普通技术人员进行进一步改进的指南,并不以任何方式构成对本发明的限制。
下述实施例中的实验方法,如无特殊说明,均为常规方法,按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例、结核分枝杆菌包涵体蛋白(如W544蛋白)复性的方法的建立
一、结核分枝杆菌蛋白包涵体工程菌的获得
1、人工合成SEQ ID NO:2所示的双链DNA分子。SEQ ID NO:2中,自5’末端起第1至6位为限制性内切酶NheI的识别位点,第7至2010位为W544基因,第2011至2016位为限制性内切酶EcoRI的识别位点。
W544基因编码氨基酸序列如SEQ ID NO:1所示的W544蛋白。W544蛋白属于结核分枝杆菌蛋白。
2、用限制性内切酶NheI和EcoRI酶切SEQ ID NO:2所示的双链DNA分子,回收酶切产物。
3、用限制性内切酶NheI和EcoRI酶切pET28a载体,回收载体骨架。
4、将酶切产物和载体骨架进行连接,得到重组质粒W544/pET28a。
将重组质粒W544/pET28a进行测序。测序结果表明,重组质粒W544/pET28a为将pET28a载体的限制性内切酶NheI和EcoRI之间的DNA小片段替换为SEQ ID NO:2自5’末端起第7至2010位所示的DNA分子,得到的重组质粒。
重组质粒W544/pET28a表达W544蛋白。
5、将重组质粒W544/pET28a导入大肠杆菌BL21(DE3),得到重组菌,即结核分枝杆菌蛋白包涵体工程菌。
二、W544蛋白的表达
1、将结核分枝杆菌蛋白包涵体工程菌的单克隆接种至5mL含50μg/ml卡那霉素的LB液体培养基,37℃、220rpm振摇培养过夜,得到菌液1。
2、将3ml菌液1接种至300ml含50μg/ml卡那霉素的LB液体培养基,37℃、220rpm振摇培养直至OD600nm为0.6-0.8,得到菌液2。
3、向步骤2得到的菌液2中加入IPTG溶液并使IPTG在体系中的浓度为0.1mM,之后16℃、150rpm诱导4h,得到菌液3。
4、分别取200μl菌液2和菌液3,室温、12000g离心5min,弃上清,用20μL 1×SDSloading缓冲液重悬菌体,100℃煮沸10min,依次得到诱导前样品和诱导后样品。
三、W544蛋白的纯化和复性
1、取步骤二中3得到的菌液3离心,收集菌体;之后PBS缓冲液重悬,得到重悬液。
2、取步骤1得到的重悬液,冰浴超声破碎10min(功率150W,破碎/间隔的时间为5s/5s),之后12000rp离心10min,收集沉淀。该沉淀即为包涵体蛋白沉淀。
3、取步骤2收集的沉淀,用结合缓冲液充分溶解,之后12000rp离心10min,收集上清液。
结合缓冲液的溶质及其浓度为50mmol/L Na2HPO4、500mmol/L NaCl、6mol/L尿素和20mmol/L咪唑,溶剂为水,pH8.0。
4、亲和层析纯化
(1)使用5倍柱体积结合缓冲液平衡层析柱,然后上样(即步骤3收集的上清液),再用10倍柱体积结合缓冲液洗涤(目的为洗涤非特异性结合蛋白),直至流出液无蛋白成分检出。
(2)用洗脱缓冲液洗脱,收集洗脱液;该洗脱液即为纯化的W544蛋白。
洗脱缓冲液的溶质及其浓度为50mmol/L Na2HPO4、500mmol/L NaCl、6mol/L尿素和500mmol/L咪唑,溶剂为水,pH8.0。
5、将1ml步骤4收集的洗脱液和1ml复性缓冲液混合,4℃静置1h;之后再加入3ml复性缓冲液,4℃静置1h,得到复性产物。
复性缓冲液的溶质及其浓度为左旋精氨酸1M、氯化钠0.2M、三羟甲基氨基甲烷0.05M、β-环状糊精1.5%、Tween200.1%,溶剂为水,pH8.0。
6、取超滤离心管(截留分子量为10000),装入复性产物,于4000rpm超滤至1.5ml左右,重新用复性缓冲液恢复至原体积,重复3次,获得的上清液即为W544蛋白溶液。
取10μl W544蛋白溶液、步骤二获得的诱导前样品和诱导后样品进行10%SDS-PAGE。
SDS-PAGE结果见图1(1为诱导前样品,2为诱导后样品,3为W544蛋白溶液,4为蛋白Marker—从上之下依次为180、130、100、70、55、40、35、25kDa)。结果表明,诱导后样品表达目的蛋白—W544蛋白,其分子量大小为80kDa;W544蛋白溶液仅含有W544蛋白一条电泳带,纯度可达到95%。
四、酶联免疫斑点法(ELISPOT)检测W544蛋白的活性
Mouse IFN-γELISPOT PLUS试剂盒为Mabtech科技有限公司的产品,code:3321-4APT-2。R4-6A2标记的单克隆抗体和链亲和素-ALP为Mouse IFN-γELISPOT PLUS试剂盒中的组件。
1、取6周、体重约为18g的BALB/c小鼠,通过尾静脉注射1.4×105cfu结核分枝杆菌,饲养观察12周,得到结核分枝杆菌感染小鼠。取6周、体重约为18g的BALB/c小鼠,饲养观察12周,得到健康小鼠。
2、完成步骤1后,在生物安全负压实验室将小鼠(结核分枝杆菌感染小鼠或健康小鼠)断颈处死,置于75%(v/v)乙醇水溶液中浸泡1-2min;之后在超净台中小心剪开小鼠的腹部外皮,再剪开腹腔,用镊子取出小鼠的脾脏。
3、完成步骤2后,取装有4-5mL 1640培养基的培养皿(规格为60mm),用镊子把尼龙网固定在平皿上,之后用注射器活塞轻轻研磨小鼠的脾脏,使脾脏细胞透过尼龙网分散于1640培养基。
4、完成步骤3后,将含有小鼠脾脏细胞的1640培养基转移至装有淋巴细胞分离液的离心管中,800g离心30min。
5、完成步骤4后,取淋巴细胞层,加入10mL 1640培养基洗涤,250g离心10min,弃上清;之后加入3-5mL含5%小牛血清的1640培养基重悬,得到重悬液;对重悬液细胞计数。
6、完成步骤5后,从Mouse IFN-γELISPOT PLUS试剂盒中取出ELISPOT板,用灭菌PBS洗板3次(200μl/孔),然后每孔加入200μl含10%小牛血清的RPMI1640培养基,室温下放置30min;移除培养基,每孔加入200μL重悬液(约含3×105个淋巴细胞),之后每孔加入20μlW544蛋白溶液(约含30μg W544蛋白);之后将ELISPOT板置于37℃、5%CO2培养箱培养24h(培养期间不可移动ELISPOT板)。
7、完成步骤6后,取所述ELISPOT板,轻轻移去ELISPOT板中的细胞,按照200μl/孔的量用PBS缓冲液洗涤5次。
8、完成步骤7后,取所述ELISPOT板,按照100μl/孔的量加入抗体稀释液,室温孵育2h。
抗体稀释液为用含0.5%FBS的PBS缓冲液将R4-6A2标记的单克隆抗体(浓度为1mg/ml)稀释至终浓度为1μg/ml得到的。
9、完成步骤8后,取所述ELISPOT板,按照200μl/孔的量用PBS缓冲液洗涤5次。
10、完成步骤9后,取所述ELISPOT板,按照100μl/孔的量加入链亲和素-ALP稀释液,室温孵育1h。
链亲和素-ALP稀释液为用含0.5%FBS的PBS缓冲液将链亲和素-ALP按照1:1000稀释获得。
11、完成步骤10后,取所述ELISPOT板,按照200μl/孔的量用PBS缓冲液洗涤5次。
12、完成步骤11后,用0.45μm过滤器过滤显色液,然后按照100μl/孔的量加入所述ELISPOT板,观察孔里斑点的变化。待斑点显色清晰后,迅速用大量自来水冲洗,拍干,避光使其自然干燥。
13、完成步骤12后,读板,扫描,统计结果。
实验重复三次,每次重复3个孔。
按照上述方法,将步骤6中的W544蛋白溶液(约含30μg W544蛋白)替换为浓度为20ug/ml的植物血凝素(PHA)溶液,其它步骤均不变,作为阳性对照。
按照上述方法,将步骤6中的W544蛋白溶液(约含30μg W544蛋白)替换为PBS缓冲液,其它步骤均不变,作为阴性对照。
检测结果见图2(A为结核分枝杆菌致病小鼠;B为健康小鼠;1为检测孔,即加入W544蛋白溶液;2为阳性对照;3为阴性对照)。结果表明,W544蛋白溶液能有效刺激结核分枝杆菌感染小鼠的脾淋巴细胞产生免疫反应,同时不刺激健康小鼠的脾淋巴细胞产生免疫反应。由此可见,复性的W544蛋白具有很好的免疫原性。
对比例1、结核分枝杆菌包涵体蛋白(如W544蛋白)复性的方法的摸索一
按照实施例中步骤一至三的方法,将步骤三中的复性缓冲液替换为复性缓冲液甲,其它步骤均不变。复性缓冲液甲的溶质及其浓度为8g NaCl、0.2g KCl、1.44g Na2HPO4和0.24g KH2PO4,溶剂为水,pH7.4。
取10μl最终获得的上清液、步骤二获得的诱导前样品、诱导后样品和复性产物超滤时的沉淀物进行10%SDS-PAGE。
SDS-PAGE结果见图3(1为诱导前样品,2为诱导后样品,3为上清液,4为复性产物超滤时的沉淀物,5为蛋白Marker—从上之下依次为180、130、100、70、55、40、35、25kDa)。结果表明,W544蛋白在复性过程中完全沉淀,上清液中没有W544蛋白存在。
对比例2、结核分枝杆菌包涵体蛋白(如W544蛋白)复性的方法的摸索二
按照实施例中步骤一至三的方法,将步骤三中的复性缓冲液替换为复性缓冲液乙,其它步骤均不变。复性缓冲液乙的溶质及其浓度为左旋精氨酸0.1M、二硫苏糖醇2mM、氯化钠0.2M、三羟甲基氨基甲烷0.05M、硫酸锌0.1mM、Tween200.3%和甘油40%,溶剂为水,pH7.4。
取10μl最终获得的上清液、步骤二获得的诱导前样品和诱导后样品进行10%SDS-PAGE。结果表明,上清液中没有W544蛋白存在。
对比例3、结核分枝杆菌包涵体蛋白(如W544蛋白)复性的方法的摸索三
按照实施例中步骤一至三的方法,将步骤三中的复性缓冲液替换为复性缓冲液丙,其它步骤均不变。复性缓冲液丙的溶质及其浓度为左旋精氨酸0.1M、二硫苏糖醇2mM、氯化钠0.2M、三羟甲基氨基甲烷0.05M、硫酸锌0.1mM、Tween200.3%和甘油40%,溶剂为水,pH9.0。
取10μl最终获得的上清液、步骤二获得的诱导前样品和诱导后样品进行10%SDS-PAGE。结果表明,上清液中没有W544蛋白存在。
对比例4、结核分枝杆菌包涵体蛋白(如W544蛋白)复性的方法的摸索四
按照实施例中步骤一至三的方法,将步骤三中5和6替换为步骤甲,其它步骤均不变。步骤甲为:将步骤4收集的洗脱液装入截留分子量为8000-14000的透析袋,用20倍复性缓冲液丁于4℃透析复性3小时以上。复性缓冲液丁的溶质及其浓度为8g NaCl、0.2g KCl、1.44g Na2HPO4和0.24g KH2PO4,溶剂为水,pH8.0。
取10μl步骤二获得的诱导前样品、诱导后样品和复性产物复性时的沉淀物和复性产物复性时的上清液进行10%SDS-PAGE。
结果表明,W544蛋白在复性过程中完全沉淀,上清液中没有W544蛋白存在。
以上对本发明进行了详述。对于本领域技术人员来说,在不脱离本发明的宗旨和范围,以及无需进行不必要的实验情况下,可在等同参数、浓度和条件下,在较宽范围内实施本发明。虽然本发明给出了特殊的实施例,应该理解为,可以对本发明作进一步的改进。总之,按本发明的原理,本申请欲包括任何变更、用途或对本发明的改进,包括脱离了本申请中已公开范围,而用本领域已知的常规技术进行的改变。按以下附带的权利要求的范围,可以进行一些基本特征的应用。
<110>中国人民解放军总医院第八医学中心
<120>一种结核分枝杆菌包涵体蛋白复性的方法及其专用复性缓冲液
<160>2
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ctgccggcaa aatttctgga aggctttgtt cgcaccagta atattaaatt tcaggatgca 720
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Claims (5)
1.一种结核分枝杆菌包涵体蛋白复性的方法,包括如下步骤:
(1)将1体积份结核分枝杆菌包涵体蛋白溶液和(0.5-1.5)体积份复性缓冲液混合,3-5℃静置0.5h-1.5h;
所述复性缓冲液的溶质及其浓度为左旋精氨酸0.8-1.2M、氯化钠0.1-0.3M、三羟甲基氨基甲烷0.03-0.07M、β-环状糊精 1.3-1.7%、Tween20 0.05-0.15%,溶剂为水,pH7.5-8.5;
(2)向完成步骤(1)的体系中加入(2.5-3.5)体积份所述复性缓冲液,混合,3-5℃静置0.5h-1.5h,得到复性产物;
(3)完成步骤(2)后,将所述复性产物装入超滤离心管,离心超滤;
(4)将完成步骤(3)的体系继续离心超滤2次以上,获得上清液;每次超滤的方法为:加入所述复性缓冲液,离心超滤;上清液即为结核分枝杆菌蛋白溶液;
所述结核分枝杆菌蛋白为W544蛋白;W544蛋白的氨基酸序列如SEQ ID NO:1所示。
2.根据权利要求1所述的方法,其特征在于:所述复性缓冲液的溶质及其浓度为左旋精氨酸1M、氯化钠0.2M、三羟甲基氨基甲烷0.05M、β-环状糊精 1.5%、Tween20 0.1%,溶剂为水,pH8.0。
3.根据权利要求1所述的方法,其特征在于:所述W544蛋白的制备方法如下:
(a1)向表达载体的多克隆位点插入编码W544蛋白的基因,得到重组质粒;
(a2)将所述重组质粒导入大肠杆菌,得到重组菌;
(a3)发酵培养所述重组菌,经破碎、收集沉淀、洗涤、离心后获得W544蛋白。
4.根据权利要求3所述的方法,其特征在于:所述编码W544蛋白的基因如SEQ ID NO:2自5’末端起第7至2010位所示。
5.权利要求1或2中所述复性缓冲液在复性结核分枝杆菌包涵体蛋白中的应用;
所述结核分枝杆菌蛋白为W544蛋白;W544蛋白的氨基酸序列如SEQ ID NO:1所示。
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