CN103088050B - 一种成熟人β防御素-2及其制备方法 - Google Patents
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Abstract
本发明公开了一种成熟人β防御素-2及其制备方法,该方法是将谷氨酸残基引入人β防御素-2的成熟序列和前肽之间,通过重组表达得到人β防御素-2前原肽,经镍柱亲和层析纯化得到较高纯度的人β防御素-2前原肽,然后用带His-tag的重组地衣芽孢杆菌谷氨酸特异性内肽酶进行酶解处理,将前肽及亲和标签切除,并再次通过亲和层析去除重组地衣芽孢杆菌谷氨酸特异性内肽酶和其他杂质,纯度较高的成熟人β防御素-2存在于穿过液中。本发明所述的成熟HBD-2制备方法具有高效、安全无毒性、成本低和纯化工艺简单的优点,从而为成熟HBD-2的大规模制备及其应用奠定基础。
Description
技术领域
本发明属于基因工程领域的基因重组表达技术领域,具体涉及一种成熟人β防御素-2的新型制备方法。
背景技术
人β防御素(HBD)是一种富含半胱氨酸的阳离子抗菌肽,对革兰氏阴性菌、革兰氏阳性菌、真菌甚至病毒都有较强的杀灭作用,因此在食品、化妆品和防腐等领域有着广泛的应用。人β防御素-2(HBD-2)于1997年在银屑病皮损组织中发现(J.Harder,J.Bartels,E.Christophers,J.Schroder,Apeptide antibiotic from human skin.Nature,1997,(387):861-861),是HBD家族成员之一,其阳离子区可以与细菌细胞膜的阴离子磷脂头部基因及水分子相互作用,形成静电力,在膜上形成电压依赖性孔道,然后插入膜中形成多聚体形成更大的孔道,从而破坏靶细胞DNA,最终导致细胞溶解(D.M.Hoover,K.R.Rajashankar,R.Blumenthal,A.Puri,J.J.Oppenheim,O.Chertov,J.Lubkowski,The structure of humanβ-defensin-2shows evidence of higherorder oligomerization.Journal of Biological Chemistry,2000,(275):32911-32918)。有研究报道人防御素对肿瘤细胞的毒性比对非肿瘤细胞的毒性要强2-50倍,而且人防御素还能显著地提高阿霉素对耐多药肿瘤细胞的裂解作用(S.Johnstone,K.Gelmon,L.Mayer,R.Hankcock,M.Bally,Peptide-mediated cytotoxicity and peptide-enhanced cytoxic activity ofdoxorubicin against wild-type and p-glycoprotein over-expressing tumor celllines,Anti-Cancer Drug Design,2000,(15):151-160),且人β防御素-2在低浓度下能趋化树突状细胞(DC)和记性性T细胞朝着受致病微生物侵袭的病灶部位迁移,能提高人体的免疫机能(Yang D,Chertov O,Bykovskaia S N,et al.Beta-defensiss:linking innate and adaptive immunity through dendriticand T cell CCR6[J].Science,1999,(287):525-552,因此HBD-2还有应用于肿瘤治疗的前景。
基于HBD-2的广泛应用前景,HBD-2在大肠杆菌细胞(X.Fang,L.Peng,Z.Xu,J.Wu,P.Cen,Cloning and expression of human beta-defensin-2gene inEseherichia coli.Protein andpeptide letters,2002,9(1):31-37)和大肠杆菌无细胞表达体系中(H.Chen,Z.Xu,N.Xu,P.Cen,Efficient production ofasoluble fusion protein containing human beta-defensin-2in E.coli cell-freesystem.Journal of biotechnology,2005,(115):307-315)均得到了表达,Fang等(X.Fang,L.Peng,Z.Xu,J.Wu,P.Cen,Cloning and expression of humanbeta-defensin-2gene in Eseherichia coli.Protein andpeptide letters,2002,9(1):31-37)在成熟HBD-2序列前融合其他分子量较大的蛋白(如硫氧还蛋白),并在两者之间引入肠激酶识别位点,所得蛋白用肠激酶进行处理从而得到成熟HBD-2,以阳离子交换层析对成熟HBD-2进行纯化;Wang等(F.Wang,X.Fang,Zhinan Xu,L.Peng,P.Cen,Fusion Expression of HumanBeta-Defensin-2from Multiple Joined Genes in Escherichia coli,PreparativeBiochemistry and Biotechnology,2003(34):215-225)在大肠杆菌中以串联形式表达HBD-2前原肽多聚体,并在成熟序列前引入甲硫氨酸,用溴化氰裂解法除去其前肽,得到成熟的HBD-2。尽管在无细胞表达体系中HBD-2的产量及纯度都较高,但其制备成本太高,不适合大规模制备;用肠激酶处理去除HBD-2的前肽,处理时间相对较长,一般需要8-16h,切割效率相对低下,而且肠激酶还存在一定程度的非特异性切割(S.H.Shahravan,X.Qu,I.S.Chan,J.A.Shin,Enhancing the specificity of the enterokinase cleavagereaction to promote efficient cleavage of a fusion tag.Protein Expression andPurification59,2008,314-319);且通过离子交换层析除去HBD-2前肽和肠激酶,无法保证成熟HBD-2的纯度;使用溴化氰裂解法除去HBD-2前肽,有较强的毒性,对实验人员健康不利,且不利于后期的应用,还存在一定的非特异性切割。
发明内容
本发明的目的在于提供一种能简单高效地获得的较高纯度的成熟HBD-2,同时提供制备上述成熟HBD-2的方法,并使其能大规模应用。
本发明的目的通过以下技术方案实现:
一种成熟人β防御素-2的制备方法,包括如下步骤:
(1)人β防御素-2前原肽重组表达载体的制备:
a.以SEQ ID NO:1-2的核苷酸序列为上、下游引物,以HBD-2前原肽序列为模板,进行PCR扩增,获得重组HBD-2前原肽目的基因序列,在其N端融合His6标签,在其C端引入终止子;所述模板是将谷氨酸残基引入HBD-2全长序列(该全长序列为密码优化过的序列,GenBank ID:AY155577),通过全合成获得重组HBD-2前原肽序列,其核苷酸序列为SEQ ID NO:3;
b.步骤a所得产物用NdeI和XhoI进行双酶切,然后与NdeI和XhoI双酶切的表达载体pET22b(+)连接,连接产物转化大肠杆菌DH5α感受态细胞;
c.从步骤b中感受态细胞中提取质粒进行双酶切及测序鉴定,所得阳性克隆转化至大肠杆菌DH5α感受态细胞中进行表达,得到HBD-2重组表达载体;
(2)重组HBD-2前原肽的制备
a.工程菌诱导表达
将步骤(1)所得HBD-2重组表达载体接种于含氨苄青霉素的液体LB培养基中,于37℃振摇过夜,然后按1:50的体积比接种于含有氨苄青霉素的液体LB培养基中,于OD=0.4-0.8时加入IPTG(异丙基硫代半乳糖苷)至终浓度为0.8mM,继续培养4h后收集菌液,离心,去除上清液,细菌沉淀用TE缓冲液洗涤2次后,用细菌裂解液重悬,于冰浴下超声,取超声后的匀浆物离心,收集上清液;
b.表达产物的纯化
取步骤a收集的上清液,利用重组表达载体在表达蛋白N端引入的6个组氨酸的标签,以Ni2+-NTA树脂亲和层析法进行纯化,即得到重组HBD-2前原肽;经过该一步纯化,其纯度可达83.7%;
(3)成熟HBD-2的制备
a.重组HBD-2前原肽的酶处理
取步骤(2)所得重组HBD-2前原肽,在20mM Tris-HCl(pH8.5)中透析12h,加入1%(w/w)的重组地衣芽孢杆菌谷氨酸特异性内肽酶(GSE-BL),于37℃处理1h后立即置于0℃冰浴中以终止反应;
b.成熟HBD-2的获得
将步骤a酶处理过的HBD-2前原肽酶解液上样至Ni2+-NTA树脂亲和层析柱,收集穿过液,成熟的HBD-2即存在于所得穿过液中。
优选地,所述的重组地衣芽孢杆菌谷氨酸特异性内肽酶的序列为SEQID NO:4所示的氨基酸序列通过基因工程方式重组表达并带有His-tag,具体制备方法见文献(W.Ye,J.Liu,H.Wang,J.Wang,X.Wang.Cloning,expression,purification,and characterization of a glutamate-specificendopeptidase from Bacillus licheniformis.Protein Expression and Purification82,2012,138–143)。
本发明与现有技术相比,具有如下优点:
(1)本发明构建的含有谷氨酸残基HBD-2前原肽重组表达载体具有构建简单,表达稳定的优点。
(2)通过亲和层析所得重组HBD-2前原肽产量为25.4mg/L,经过重组地衣芽孢杆菌谷氨酸特异性内肽酶处理,HBD-2前肽被去除,再经过亲和层析得到纯度较高的成熟HBD-2,其产量为23.2mg/L,纯度为97.9%。该去除HBD-2前肽的方法具有催化效率高、酶切作用特异性强、收率高的优点。
(3)重组地衣芽孢杆菌谷氨酸特异性内肽酶也具有亲和标签,因此便于通过亲和层析进行去除。
(4)该重组地衣芽孢杆菌谷氨酸特异性内肽酶利用基因工程方法重组表达,来源方便,因此能降低成熟HBD-2的生产成本。
鉴于HBD-2全长序列中不存在谷氨酸,因此我们可以在HBD-2前肽和其成熟序列间引入谷氨酸位点,再用底物专一性较强且对谷氨酸具有较高催化效率的重组地衣芽孢杆菌谷氨酸特异性内肽酶(带His-Tag)去除其前肽,并经亲和层析纯化得到纯度较高的成熟HBD-2。本发明所述的成熟HBD-2制备方法具有高效、安全无毒性、成本低和纯化工艺简单的优点,从而为成熟HBD-2的大规模制备及其应用奠定基础。
附图说明
图1为HBD-2前原肽重组质粒构建图;
图2为重组菌双酶切鉴定图,其中:泳道M:DNA marker,泳道1:HBD-2前原肽重组质粒双酶切;泳道2:表达载体pET-22b(+)双酶切产物;
图3为HBD-2前原肽重组质粒的测序结果图;
图4为重组HBD-2前原肽表达的Tricine-SDS-PAGE鉴定的电泳图;其中:泳道1:未诱导总菌;泳道2:OD0.4诱导上清;泳道3:OD0.4诱导沉淀;泳道4:OD0.6诱导上清;泳道5:OD0.6诱导沉淀;泳道6:OD0.8诱导上清;泳道7:OD0.8诱导沉淀;泳道9:unstained protein Marker;
图5为重组HBD-2前原肽纯化及其前肽去除的Tricine-SDS-PAGE鉴定的电泳图;其中:泳道1:未诱导总菌;泳道2:诱导后上清;泳道3:200mM咪唑洗脱液;泳道4:洗脱液透析后GSE-BL处理15min;泳道5:洗脱液透析后经GSE-BL处理1h;泳道6:处理1h后上Ni柱穿过液;泳道M:unstained protein Marker;
图6为重组HBD-2前原肽纯化及其前肽去除的Western blot鉴定的电泳图;其中:泳道1:未诱导总菌;泳道2:200mM咪唑洗脱液;泳道3:洗脱液透析后GSE-BL处理15min;泳道4:洗脱液透析后经GSE-BL处理1h;泳道5:处理1h后上Ni柱穿过液;
图7为成熟HBD-2对铜绿假单胞菌和金黄色葡萄球菌的抑菌效应图;
图8为成熟HBD-2的细胞毒性检测结果图。
具体实施方式
本发明采用分子生物学方法,扩增得到含有谷氨酸残基的重组人β防御素-2(HBD-2)前原肽编码DNA序列,将此DNA序列克隆入原核表达载体中,构建重组表达载体,并转化入宿主菌中进行表达,用Ni2+柱进行亲和层析纯化后得到重组HBD-2前原肽,然后用带组氨酸标签的地衣芽孢杆菌谷氨酸特异性内肽酶进行处理,再上样至Ni2+柱进行亲和层析,即可获得高纯度的成熟HBD-2,其抑菌实验和细胞毒性检测结果进一步证实了所得成熟HBD-2具备生物活性。
可用于表达本发明中的重组HBD-2前原肽的宿主没有特别限制,代表性例子包括大肠杆菌BL21、毕赤酵母KM71和哺乳动物细胞COS7等多种宿主。大肠杆菌BL21(DE3)是Lon蛋白酶和Omp T蛋白酶缺陷型,可保持目的蛋白不被降解。且使用大肠杆菌BL21(DE3)进行表达具有操作简便、成本低、周期短和表达量高等优点,因此本发明优选大肠杆菌BL21(DE3)作为表达宿主。
以下将参照附图,对本发明的优选实施例进行详细的描述。优选实施例中未注明具体条件的实验方法,通常按照常规条件,例如分子克隆实验指南(第三版,J.萨姆布鲁克等著,黄培堂等译,科学出版社,2002年)中所述条件,或按照制造厂商所建议的条件进行操作。
实施例1
一、产重组人β防御素-2前原肽工程菌的构建
通过在编码人β防御素-2前肽和成熟序列之间引入谷氨酸残基作为谷氨酸特异性内肽酶的识别位点,并在N端融合组氨酸标签,C端引入终止子,经NdeI和XhoI双酶切后与表达载体pET22b(+)连接,即可实现重组质粒pET22b-HBD-2前原肽的构建,其具体构建过程如图1所示。
1、目的基因片段、引物的设计与合成
根据人β防御素-2基因序列(GenBank ID:AY155577),在其成熟序列基因和其前肽基因片段之间引入GAA作为谷氨酸密码子,新的重组HBD-2前原肽基因序列(见SEQ ID NO:3)由上海捷瑞生物工程有限公司合成,并根据此序列设计引物,其中正向引物为SEQ ID NO:1-5’-GGAATTCCATATG ATGCGTGTTCTGTATCTG-3’,含酶切位点NdeI(下划线标记部分),斜线部分为编码N端组氨酸标签的核苷酸序列,反向引物为SEQ ID NO:2-5’-CCGCTCGAGTTATGGCTTCTTACAGCACTTGGTGCCT-3’,含有酶切位点XhoI(下划线标记部分)。
2.PCR扩增及重组质粒构建
以合成的HBD-2前原肽基因序列(见SEQ ID NO:3)为模板,以SEQID NO:1-2所示的核苷酸序列为上、下游引物进行PCR扩增,其反应体系如下:
PCR扩增程序如下:
所得PCR产物于4℃保存待用,1%琼脂糖凝胶电泳鉴定PCR产物,得到大小为238bp的包含酶切位点、N端组氨标签和HBD-2前原肽目的基因的片段,对此片段切胶回收。
所得PCR产物用NdeI和XhoI于37℃双酶切5h,pET-22b(+)(购于Novagen公司)也用NdeI和XhoI于37℃双酶切3h。双酶切体系如下:
切胶回收双酶切产物,于16℃连接10h,连接体系如下:
所得连接产物转化大肠杆菌(Escherichia coli)DH5α感受态细胞(购于Invitrogen公司)。
3.重组质粒鉴定
方法:上述连接产物转化大肠杆菌DH5α感受态细胞后,挑取9个单克隆扩大培养,并进行菌液PCR鉴定,PCR条件同步骤2,选取经菌液PCR鉴定为阳性的克隆提取质粒,NdeI和XhoI37℃双酶切2h,双酶切产物进行琼脂糖凝胶电泳鉴定。选择相应的阳性克隆送样至华大基因进行测序鉴定。
结果:如图2所示,重组质粒双酶切后得到与pET-22b(+)大小一致的大片段和与HBD-2前原肽目的基因大小一致的小片段。证明此克隆为阳性克隆,其测序结果如图3所示,与SEQ ID NO:3所示的核苷酸序列完全一致。因此,HBD-2前原肽重组质粒成功构建。
二、人β防御素-2前原肽的表达、纯化及成熟化
1.人β防御素-2前原肽的表达
方法:上述实施例1中的阳性克隆提取得到的质粒转化至大肠杆菌感受态细胞BL21(DE3)(购于Invitrogen公司)中,挑取经双酶切鉴定的阳性克隆接种至3mL含有100μg/mL Amp的LB培养基中扩大培养,于OD600分别为0.4、0.6和0.8时加入0.8mM IPTG于37℃诱导表达,OD600为2.0时离心收集菌体,超声裂解,上清和沉淀分别加入上样缓冲液沸水煮5min。浓缩胶60V,间隙胶100V,分离胶120V进行Tricine-SDS-PAGE电泳,电泳完毕用考马斯亮蓝染色液染色,脱色液脱色。
结果:如图4所示,重组HBD-2前原肽得到成功诱导表达,其分子量大约为10.2kD,而其理论分子量为8.02kD。可能是N端的组氨酸标签所带电荷影响了其电泳行为,使其表观分子量比理论分子量要大。在OD为0.4时进行诱导裂解后所得上清中重组HBD-2前原肽表达量最高,占上清中总蛋白的19.7%,而OD为0.6时重组HBD-2前原肽占上清中总蛋白的13.6%,OD为0.8时重组HBD-2前原肽占上清中总蛋白的10.9%。
结论:OD600为0.4时加入IPTG诱导可在裂解后所得上清液中获得更多的重组HBD-2前原肽,因此选择OD600为0.4时加入IPTG进行诱导表达。
2、人β防御素-2前原肽的纯化
方法:将上述阳性克隆接种至10mL含有100μg/mL Amp的LB培养基中扩大培养12h,然后以2%的接种量接种至450mL LB液体培养基中,于OD600为0.4时加入IPTG诱导表达。37℃诱导4h后,12000rpm离心5min,弃去上清,然后用TE buffer洗涤菌体,12000rpm离心5min,弃去上清,对菌体进行称重,然后加入裂解液(20mM Tris-HCl,500mM NaCl,1mM PMSF,pH8.0)进行超声破碎,每克湿菌体加入10mL裂解液。
超声结束后于4℃,12000×g离心20min,所得上清上样至Ni2+-NTA亲和层析柱(HisTrap,HP1mL,GE公司),然后用含有咪唑的洗脱液进行洗脱。
结果:如图5所示,人β防御素-2前原肽得到成功诱导表达,经过亲和层析后,在含有200mM咪唑的洗脱液中得到重组HBD-2前原肽,其纯度为83.7%。
结论:经过亲和层析可得到纯度较高的重组人β防御素-2前原肽。
3、人β防御素-2前原肽的成熟化及纯化
方法:对步骤2中得到的200mM咪唑洗脱液用20mM Tris-HCl(pH8.5)进行透析,然后加入1%(w/w)的重组地衣芽孢杆菌谷氨酸特异性内肽酶(GSE-BL)于37℃处理1h,处理结束后立即置于0℃以终止反应。上述GSE-BL处理过的产物上样至Ni-NTA亲和层析柱,收集穿过液,上样结束后用20mM Tris-HCl(pH8.5)平衡,然后用含有咪唑的洗脱液进行洗脱。
结果:如图5所示,HBD-2前原肽经重组GSE-BL处理后可得到分子量大约为4.3kD的片段,其分子量与成熟HBD-2肽段大小一致(图5,lane5)。重组HBD-2前原肽经GSE-BL处理15min后,只有19.6%的HBD-2前原肽的前肽被去除(图5,lane4);GSE-BL处理1h后,94.6%的HBD-2前原肽的前肽被去除。处理1h的样品上样至Ni柱,大部分蛋白以穿过液形式流出,用含有咪唑的洗脱液进行洗脱,没有洗脱峰出现,极少部分未去除标签的HBD-2前原肽、重组GSE-BL及其他杂质由于带组氨酸标签而与Ni柱结合,从而与成熟HBD-2得到分离(图5,lane7),所得成熟HBD-2纯度为97.9%。重组HBD-2前原肽的产量为25.4mg/L,成熟HBD-2的产量23.2mg/L。
结论:HBD-2经过重组GSE-BL处理1h几乎可以去除所有重组HBD-2前原肽的前肽,再经过亲和层析纯化后,成熟HBD-2的纯度进一步提高。
4.人β防御素-2前原肽的Western blot鉴定
方法:HBD-2表达、纯化及成熟化的样品经Tricine-SDS-PAGE电泳后,转移至PVDF膜上。转膜电压为100V,转膜时间为30min。转膜结束后,将膜于甲醇中固定1min,再用超纯水冲洗,晾干数分钟。然后将膜置于5%脱脂奶粉中,于室温振摇3h,封闭结束后,将膜置于用5%脱脂奶粉稀释1500倍的鼠源的Anti-His Tag单克隆抗体(Abcam公司,英国)中,37℃温育1h,用TBST充分洗涤后加入发光工作液(Thermo scientific公司,美国),于暗室中显影曝光。
结果:如图6所示,未诱导样品无条带,200mM咪唑洗脱液在10kD附近呈现较明显的条带(图6,lane2),HBD-2前原肽经GSE-BL处理15min后,10kD左右条带有所减弱,生成的新片段不带有组氨酸标签(图6,lane3);经GSE-BL处理1h后,10kD左右条带几乎完全消失;GSE-BL处理过的样品经过Ni2+柱后,带组氨酸标签的杂质与成熟的HBD-2完全分离(图6,lane5)。
结论:重组GSE-BL处理1h几乎可以去除所有重组HBD-2的前肽,所得成熟HBD-2中不含有其他带组氨酸标签的杂质。
三、成熟人β防御素-2生物活性检测
1、成熟人β防御素-2对致病菌的抑制作用
方法:取不同浓度(0、25μg/mL、50μg/mL、100μg/mL和200μg/mL)的成熟HBD-2加入培养16h的金黄色葡萄球菌及铜绿假单胞菌的菌液中,12h后取样,测量菌液于600nm的OD值,以确定得到成熟HBD-2对不同细菌的抑制率。
结果:如图7所示,25μg/mL的成熟HBD-2对铜绿假单胞菌的抑制率为52.0±0.72%,而200μg/mL的成熟HBD-2对于金黄色葡萄球菌的抑制率为52.5±1.8%。
结论:制备得到的成熟HBD-2对于铜绿假单胞菌的抑菌效果比对金黄色葡萄球菌的抑菌效果要显著。
2.成熟人β防御素-2的细胞毒性
A.HCT-8细胞的培养
方法:取出冻存管,用吸管吸出人结肠癌细胞HCT-8细胞悬液,于含有15%胎牛血清(FCS)的RPMl1640培养基中培养,置于37℃,5%的CO2培养箱中培养,每3天换液一次。实验时取对数生长期细胞,混匀后计数。
B.小鼠骨髓DC细胞提取
方法:小鼠禁水禁食12h,摘眼放血后断颈处死,于超净工作台中无菌开腹,取出股骨和肱骨,放入已盛有5mL PBS的平皿中清洗。用1mL无菌注射器针头插入腿骨,冲出骨髓细胞,红细胞裂解液裂解2分钟。1000×g离心3min,弃去上清,用RPMl1640清洗两遍,然后加入含有GM-CSF和IL-4的细胞培养液培养DC细胞。一般培养2天-4天待其形成集落后再进行实验。
C.成熟人β防御素-2细胞毒性检测
方法:取对数生长期的人结肠癌细胞HCT-8,先用0.25%胰酶消化,再用含有15%胎牛血清(FCS)的RPMI1640培养液制备成单细胞悬液,计数后以1×105个/mL,100μL/孔接种于96孔培养板中,细胞培养过夜。试验孔分别加入终浓度为0、10μg/mL、25μg/mL、50μg/mL和100μg/mL的成熟HBD-2。阴性对照组中仅加细胞不加药物,空白对照组中不加药物和细胞,各组设3个平行孔。37℃,5%CO2条件下培养72h后,每孔加入MTT10μL(5mg/mL),37℃下再培养4h后终止培养,小心吸弃孔内培养液上清。加入DMSO150μL/孔,震荡15min,使结晶物充分溶解。选择490nm波长,在酶标仪上测各孔吸光度(OD值)。取每3个平行孔OD值的平均数,计算加入样品对细胞的抑制率。
结果:成熟HBD-2的细胞毒性检测结果如图8所示,10μg/mLHBD-2对于HCT-8的抑制率是对于小鼠骨髓DC细胞抑制率的1.8倍,100μg/mLHBD-2对于HCT-8的抑制率是对于小鼠骨髓DC细胞抑制率的3.3倍。
结论:制备得到的成熟HBD-2对于人结肠癌细胞HCT-8的毒性要强于对小鼠骨髓DC细胞的毒性。因此HBD-2具有广阔的抗肿瘤应用前景。
序列表
<110>华南理工大学
<120>一种成熟人β防御素-2及其制备方法
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<170>PatentIn version3.3
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<212>DNA
<213>人工序列
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<223>引物
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<211>37
<212>DNA
<213>人工序列
<220>
<223>引物
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ccgctcgagt tatggcttct tacagcactt ggtgcct 37
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<211>195
<212>DNA
<213>人工序列
<220>
<223>HBD-2前原肽序列
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gtattcggtg aaggcattgg tgatccagtc acctgcctga aatccggtgc catctgtcat 120
cctgtgttct gtcctcgtcg ttacaagcaa attggtactt gcggtttgcc aggcaccaag 180
tgctgtaaga agcca 195
<210>4
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<212>PRT
<213>地衣芽孢杆菌(Bacillus licheniformis)ATCC14580
<220>
<223>重组地衣芽孢杆菌谷氨酸特异性内肽酶的氨基酸序列
<400>4
Ser Val Ile Gly Ser Asp Asp Arg Thr Arg Val Thr Asn Thr Thr
1 5 10 15
Ala Tyr Pro Tyr Arg Ala Ile Val His Ile Ser Ser Ser Ile Gly
20 25 30
Ser Cys Thr Gly Trp Met Ile Gly Pro Lys Thr Val Ala Thr Ala
35 40 45
Gly His Cys Ile Tyr Asp Thr Ser Ser Gly Ser Phe Ala Gly Thr
50 55 60
Ala Thr Val Ser Pro Gly Arg Asn Gly Thr Ser Tyr Pro Tyr Gly
65 70 75
Ser Val Lys Ser Thr Arg Tyr Phe Ile Pro Ser Gly Trp Arg Ser
80 85 90
Gly Asn Thr Asn Tyr Asp Tyr Gly Ala Ile Glu Leu Ser Glu Pro
95 100 105
Ile Gly Asn Thr Val Gly Tyr Phe Gly Tyr Ser Tyr Thr Thr Ser
110 115 120
Ser Leu Val Gly Thr Thr Val Thr Ile Ser Gly Tyr Pro Gly Asp
125 130 135
Lys Thr Ala Gly Thr Gln Trp Gln His Ser Gly Pro Ile Ala Ile
140 145 150
Ser Glu Thr Tyr Lys Leu Gln Tyr Ala Met Asp Thr Tyr Gly Gly
155 160 165
Gln Ser Gly Ser Pro Val Phe Glu Gln Ser Ser Ser Arg Thr Asn
170 175 180
Cys Ser Gly Pro Cys Ser Leu Ala Val His Thr Asn Gly Val Tyr
185 190 195
Gly Gly Ser Ser Tyr Asn Arg Gly Thr Arg Ile Thr Lys Glu Val
200 205 210
Phe Asp Asn Leu Thr Asn Trp Lys Asn Ser Ala Gln Leu Glu His
215 220 225
His His His His His
230
Claims (2)
1.一种成熟人β防御素-2的制备方法,其特征在于,包括如下步骤:
(1)人β防御素-2前原肽重组表达载体的制备
a.以SEQ ID NO:1-2的核苷酸序列为上、下游引物,以HBD-2前原肽序列为模板,进行PCR扩增,获得重组HBD-2前原肽目的基因序列,在其N端融合His6标签,在其C端引入终止子;所述模板的核苷酸序列为SEQ ID NO:3;
b.步骤a所得产物用NdeI和XhoI进行双酶切,然后与NdeI和XhoI双酶切的表达载体pET22b连接,连接产物转化大肠杆菌DH5α感受态细胞;
c.从步骤b中感受态细胞中提取质粒进行双酶切及测序鉴定,所得阳性克隆转化至大肠杆菌DH5α感受态细胞中进行表达,得到HBD-2重组表达载体;
(2)重组HBD-2前原肽的制备
a.工程菌诱导表达
将步骤(1)所得HBD-2重组表达载体接种于含氨苄青霉素的液体LB培养基中,于37℃振摇过夜,然后按1:50的体积比接种于含有氨苄青霉素的液体LB培养基中,于OD=0.4-0.8时加入IPTG至终浓度为0.8mM,继续培养4h后收集菌液,离心,去除上清液,细菌沉淀用TE缓冲液洗涤2次后,用细菌裂解液重悬,于冰浴下超声,取超声后的匀浆物离心,收集上清液;
b.表达产物的纯化
取步骤a收集的上清液,以Ni2+-NTA树脂亲和层析法进行纯化,即得到重组HBD-2前原肽;
(3)成熟HBD-2的制备
a.重组HBD-2前原肽的酶处理
取步骤(2)所得重组HBD-2前原肽,在20mM、pH8.5的Tris-HCl中透析12h,加入1%w/w的重组地衣芽孢杆菌谷氨酸特异性内肽酶,于37℃处理1h后立即置于0℃冰浴中以终止反应;
b.成熟HBD-2的获得
将步骤a酶处理过的HBD-2前原肽酶解液上样至Ni2+-NTA树脂亲和层析柱,收集穿过液,成熟的HBD-2即存在于所得穿过液中。
2.根据权利要求1所述的制备方法,其特征在于,所述的重组地衣芽孢杆菌谷氨酸特异性内肽酶的序列为SEQ ID NO:4。
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