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1、 人杀菌渗透增强性蛋白N末端片段在大肠杆菌中的表达 作者：董伟，董晓慧，楚雍烈，杨娥，胡刚，郑建武 【关键词】 基因表达 摘要：目的 构建表达人杀菌渗透增强性蛋白(BPI) N端193个氨基酸的重组表达质粒，诱导表达BPI193重组蛋白。方法 应用RTPCR的方法从HL60细胞内扩增出BPI氨基端1-193个氨基酸的基因序列，克隆入T载体。将BP
2、I193基因片段定向克隆入原核表达载体PET28a中，构建重组的原核表达质粒PETBPI193，转化大肠杆菌BL21菌株，用IPTG诱导表达蛋白。应用SDSPAGE检测蛋白表达情况，计算机薄层扫描分析蛋白占菌体总蛋白百分比；Westernblot技术检测表达蛋白的特异性。结果 从HL60细胞中扩增得到579bp的BPI193基因片段，构建了TBPI193亚克隆和PETBPI193重组表达质粒。转化大肠杆菌BL21，通过IPTG诱导，经SDSPAGE检测表明，成功表达了6HisBPI193融合蛋白。计算机薄层扫描分析表达量占菌体总蛋白的12%。Westernblot检测显示表达产物具
3、有特异性。结论 成功构建了PETBPI193重组表达质粒。在大肠杆菌BL21内，诱导获得了BPI193与组氨酸融合表达的重组蛋白。关键词：杀菌/渗透增强性蛋白；基因表达；大肠杆菌Expression of human bactericidal/permeability increasing proteinNterminal fragment in E.coliABSTRACT: Objective To construct a prokaryotic expression plasmid of cDNA sequence encoding first 193 amino
4、 acids of BPI Nterminal and to express it in E.coli BL21. Methods Total RNA was extracted from HL60 cell and then was amplified by using RTPCR. The PCR product was cloned into pMD18T vector. BPI193 gene was directly cloned into PET28a plasmid on the role of T4 DNA ligase. PETBPI193 recombinant
5、 expression vector was transformed into competent E.coli BL21 and protein expression was induced by IPTG. Fusion protein was detected by SDSPAGE and Westernblot. Results A 579bp DNA amplicon was amplified by RTPCR. TBPI193 subclone and PETBPI193 recombinant expression plasmid was constructed s
6、uccessfully. 6HisBPI193 fusion protein containing 193 amino acids of BPI Nterminal was expressed by IPTG induced method. Fusion protein was detected by SDSPAGE and confirmed by Westernblot. The expressed fusion protein constituted 12% of total bacterial protein. Conclusion PETBPI193 recombinan
7、t expression plasmid was constructed successfully. To transform PETBPI193 recombinant plasmid into E.coli BL21 and 6HisBPI193 fusion protein was expressed by IPTG induced manner.KEY WORDS: bactericidal/permeability increasing protein; gene expression; E.coli迄今为止，对脂多糖引起的并发症，如内毒素血症与败血症休克仍没有有效的治疗手段，临床治
8、疗急需新的抗革兰阴性(G-)菌感染的药物。人杀菌渗透增强性蛋白(bactericidal/permeability increasing protein ，BPI)是对细菌高度稳定的非催化蛋白，主要存在于人及哺乳动物多形核白细胞(PMN)的嗜天青颗粒中,也可微量表达于PMN和单核细胞表面13。最新研究表明，BPI还可表达于嗜酸性粒细胞和上皮细胞45，它不仅具有特异性杀伤G-菌的作用，还可以中和内毒素，是一种具有广阔应用前景的“新型抗生素”6。在本实验中，我们建立了稳定表达BPI氨基端(BPI193)的原核表达系统，获得了高产量的纯化蛋白，为进一步研究BPI在体内外的生物活性，研发新的BPI产品
9、创造条件。1 材料与方法1.1 细胞株、菌种和质粒 HL60细胞株由第四军医大学微生物教研室雷迎峰博士惠赠；E.coli DH5和E.coli BL21为本室保存菌株；pMD18T 载体为Takara公司产品；PET28a原核表达质粒为Novagen公司产品。1.2 主要试剂 Trizol为Invitrogen公司产品；cDNA第一链合成逆转录试剂盒购自Fermentas公司；Xho、BamH限制性内切酶、TaqDNA 聚合酶、T4DNA连接酶均为Takara公司产品；DEPC为Sigma公司产品；DNTPs购自北京鼎国生物公司；小剂
10、量质粒提取试剂盒和DL2000 DNA marker购自北京天为时代公司，小分子量蛋白marker购自华美公司；溃疡性结肠炎患者血清由西安交通大学医学院第二附属医院检验科李步荣医师惠赠；山羊抗人HRPIgG购自博士德生物公司；1640培养基购自Gibco公司；DNA凝胶回收试剂盒为北京赛百盛公司产品。1.3 1引物的设计与合成 根据GenBank数据库已收录的BPI cDNA的基因序列，借助于软件Primer Premier 5.0和DNA club设计了扩增BPI 1193个氨基酸的上下游扩增引物。P1 5 CTGGATCCATGGTCAACCCTGGCG
11、TCGT 3(BamH)P2 5 CCGCTCGAGTTACAGAGTCTGGAAATAAGG 3(Xho) 在引物的5端分别添加了BamH和Xho两个酶切位点，以确保酶切后可以插入PET28a原核表达载体中，并且读码框正确，还添加了起始密码ATG和终止子TAA。扩增的BPI位于124-702bp，DNA长度为579bp。引物由北京三博远志生物技术有限责任公司合成。1.4 方法1.4.1 RTPCR扩增目的基因 用Trizol试剂从HL60细胞中提取总RNA，按试剂盒说明反转录出cDNA
12、第一链，然后进行PCR扩增目的基因。经15g/L的琼脂糖凝胶电泳分析结果后，用凝胶回收试剂盒回收PCR产物。通过10g/L的琼脂糖凝胶电泳观察回收情况并目测定量。1.4.2 PCR产物与T载体的连接及鉴定 按照pMD18T载体说明书， 进行PCR回收产物和pMD18T载体的连接反应。将连接产物转化感受态E.coli DH5，经抗生素和蓝白斑筛选出阳性菌落，然后用PCR、限制性内切酶分析和DNA测序证实BPI193是否插入T载体以及基因序列有无突变。1.4.3 PETBPI193重组质粒的构建 分别用BamH和Xho双酶切TBPI193和原核表达载
13、体PET28a，酶切产物经10g/L琼脂糖凝胶电泳后，用北京赛百盛公司凝胶回收试剂盒回收，DNA定量后在T4DNA连接酶的作用下将BPI193定向克隆入原核表达载体PET28a中，构建重组的原核表达质粒PETBPI193。转化大肠杆菌BL21菌株，筛选出阳性菌落，并采用PCR和限制性酶切技术鉴定，得到表达菌。1.4.4 目的蛋白的诱导表达和鉴定 用IPTG诱导表达菌，采用不同的IPTG诱导浓度(1、2、3、4mmol/L)和诱导时间(1、2、3、4、5、6h)，应用SDSPAGE技术检测细菌蛋白表达情况，获得最佳表达条件，计算机薄层扫描分析表达目的蛋白占菌体总蛋白百分比
14、。应用WesternBlot技术检测表达蛋白的特异性。2 结果2.1 RTPCR扩增结果 RTPCR反应后取5L经15g/L琼脂糖凝胶电泳检测，结果扩增出约602bp的基因片段，与预期大小相符。2.2 BPI193基因片段与T载体的连接 将获得的BPI193基因片段RTPCR的产物凝胶回收后，按照pMD18T载体说明书操作，双酶切结果切出约585bp和2690bp左右的基因片段，与预期相符(图1)。送上海华诺公司测序，证明BPI193基因序列完全正确，命名为TBPI193。图1 重组质粒TBPI193的PCR、双酶切鉴定（
15、略）Fig.1 Identification of plasmid TBPI193 by PCR and restriction enzyme digestion1: DL2000 DNA marker; 2: BPI193 from PCR; 3: restriction enzyme digestion with BamH and Xho2.3 PETBPI193重组质粒的构建 用BamH和Xho分别双酶切TBPI193和PET28a质粒，凝胶回收试剂盒回收得到纯化产物。用T4连接酶连接，转化E.coli DH5，卡那霉素筛选细菌，并进行PCR和BamHXho双酶切
16、鉴定，PCR扩增出约602bp的基因片段，双酶切获得约585bp和5329bp的基因片段，与预期相符(图2)。图2 PETBPI193重组质粒的PCR、酶切鉴定（略）Fig.2 Identification of recombinant plasmid PETBPI193 by PCR and restriction enzyme digestion1: DL2000DNA marker; 2: PCR product of TBPI193; 3:PCR product of PETBPI193; 4: double restriction enzyme digestion wit
17、h BamH and Xho2.4 BPI193重组蛋白的表达和鉴定 用IPTG诱导表达菌BL21，发现随时间延长蛋白量有所增加，在4h时表达量较大。在不同IPTG浓度的条件下诱导蛋白表达， 2-3mmol/L时蛋白表达量比较丰富。诱导后在21ku左右处有蛋白表达，该蛋白大小与预期相符。经薄层扫描分析，表达蛋白量占菌体总量的12%左右。可溶性分析表明表达的BPI193重组蛋白以包涵体的形式释放(图3)。Westernblot鉴定，结果表明用IPTG诱导后的PETBPI193表达菌在相应约21ku位置出现特异性染色带，而未诱导的表达菌没有该条带(图4)，说明表达的目的蛋白
18、具有特异性。图3 重组蛋白BPI193在IPTG诱导浓度不同时的表达情况（略）Fig.3 Expression of recombinant protein BPI193 at different inducer concentrations1: lower MW protein marker; 2: noninduced PETBPI193; 3-6: IPTG induced PETBPI193 at 1, 2, 3, 4mmol/L图4 重组蛋白BPI193的Westernblot鉴定（略）Fig.4 Identification of recombinant p
19、rotein BPI193 by Westernblot1: noninduced PETBPI193; 2: induced PETBPI193 3 讨论根据BPI氨基端的基因序列设计了1对特异性引物，采用RTPCR的方法从HL60细胞中成功扩增了602bp的基因片段，与预期的目的基因片段大小相符。将BPI193基因片段克隆入pMD18T载体，构建TBPI193重组质粒。转化E.coli DH5，通过蓝白斑筛选获得阳性克隆，经PCR和限制性内切酶鉴定分析(BamH和Xho双酶切)，均获得了与预期相符的实验结果。经DNA测序分析，本实验获得的BPI193基因序列与文献报道完全一致，
20、没有突变。PET28a为原核表达载体。将BPI193基因片段定向克隆入PET28a原核表达载体中，转化大肠杆菌DH5，初步筛选出阳性克隆后，通过菌落PCR以及BamH和Xho双酶切分析进一步鉴定，与预期结果相一致。最后再进行DNA测序分析，确证BPI193基因片段准确无误的正向插入到表达载体中，并且BPI193的N末端与6个组氨酸相连接。PETBPI193重组表达质粒构建成功。PETBPI193重组表达质粒转化表达菌E.coli BL21，经过IPTG诱导，获得了以包涵体形式表达的预期重组蛋白，其分子质量为21ku左右，并且可以与BPI抗体特异性结合，充分证明BPI193表达成功。传统制备BP
21、I多采用柱层析的方法，国外获得BPI N端功能性片段多采用真核系统表达611，但这些方法生产条件要求比较高，消耗巨大，影响了临床应用。研究表明BPI糖基化部位与蛋白的活性没有显著关系，BPI是否糖基化对于抗体与抗原的识别来说没有明显影响12。本研究采用原核表达系统表达BPI193重组蛋白，大大降低了生产成本，为后续的实验研究奠定了基础。本研究选用PET28a作为原核表达载体，它含有T7启动子，是一种T7噬菌体表达系统，具有能转录大肠杆菌RNA聚合酶不能有效转录基因的优点。其多克隆位点上游有6个组氨酸和凝血酶位点，可利用固化Ni2+亲和层析柱纯化表达的融合蛋白，还可以用凝血酶切割纯化的融合蛋白，
22、从而获得重组的目的蛋白。是一种较好的表达载体。为了获得最优化的蛋白表达条件，在IPTG终浓度为1mmol/L时延长表达时间，发现随时间延长蛋白量有所增加，但增长幅度不大，在4h时表达量较大。在不同IPTG浓度的条件下诱导蛋白表达，发现2-3mmol/L时蛋白表达量比较丰富。生长温度可能是影响大肠杆菌高水平表达的主要因素之一，通过试验确定最佳温度是表达外源蛋白的关键。本实验分别在加入IPTG后于37、35、33进行BPI193重组蛋白的诱导表达，虽然几种温度条件下融合蛋白都可以表达，但温度较低时蛋白表达量更加丰富，表达也比较稳定。BPI193基因片段被定向克隆于PET28a原核表达载体后，其N端
23、与6个组氨酸形成融合蛋白，并在IPTG的诱导下得以表达。经蛋白的可溶性分析显示，BPI193与组氨酸融合蛋白在细菌中主要以包涵体的形式进行表达，而在细菌裂解液上清中没有检测到BPI193融合蛋白。虽然反复优化诱导表达条件，但BPI193重组蛋白总是以包涵体的形式存在。文献中也提示，几种利用原核表达系统表达人BPI时，目的蛋白也总是以包涵体的形式释放。我们推测可能与以下因素有关：大肠杆菌BL21进入对数生长期时间短，生长速度快，由于细菌生长过度或者生长过速加重细菌合成系统的负担，导致形成包涵体；BPI193是BPI N末端前193个氨基酸，包含了3个与生物活性密切相关的区域，具有完整
24、BPI特异性杀伤GNB、结合及中和LPS的能力。大肠杆菌本身就是革兰阴性菌，因此对于E.coli BL21来说，BPI193重组蛋白具有细胞毒性，可能会对细菌产生杀伤作用。所以细菌在合成过程中将蛋白纳入包涵体中，消除或减弱蛋白的生物活性，减轻对宿主菌的毒性作用。BPI193重组蛋白以包涵体的形式存在有助于表达产物的分离和提取，也可以减少胞内蛋白酶对蛋白的破坏和降解。因此，在本实验中具有有利的方面。但是，要获得具有功能性的重组蛋白还需要进行复性处理，回复蛋白的空间结构和生物活性，这会给生产使用带来一些麻烦，有待进一步完善。虽然经过多种方法优化蛋白表达条件(包括延长诱导时间，增加诱导剂浓度，降低诱
25、导时细菌培养温度)，但是BPI193融合蛋白的表达量仍然很低，经分析只占菌体总蛋白的12%左右。这可能是因为：诱导条件没有达到最优，诱导前后细菌培养的条件不合适；Bluow等13研究发现，完整的BPI以及含有BPI C端和LBP N端的嵌合体都可以稳定的储存在细胞中，而缺失了C端的BPI在储存过程中会被降解。所以，推测部分BPI193与组氨酸的融合蛋白在表达过程中可能被细菌降解。这尚需在今后的实验中进一步研究。参考文献：1Weersink AJ, van Kessel KP, van den Tol ME, et al. Human granulocytes express a 55kDa l
26、ipopolysaccharidebinding protein on the cell surface that is identical to the bactericidal/permeabilityincreasing protein J. J Immunol, 1993,150(1):253263.2Weiss J. Leucocytederived antimicrobial proteins J. Curr Opin Hematol, 1994,1(1):7884.3Calafat J, Janssen H, Knol EF, et al. The bactericidal/pe
27、rmeabilityincreasing protein (BPI) is membraneassociated in azurophil granules of human neutrophils, and relocation occurs upon cellular activation J. APMIS, 2000,108(3):201208.4Calafat J, Janssen H, Tool A, et al. The bactericidal/ permeabilityincreasing protein (BPI) is present in specific granule
28、s of human eosinophils J. Blood, 1998, 91(12):47704775.5Geraldine Canny, Ofer Levy, Glenn TF, et al. Lipid mediatorinduced expression of bactericidal permeabilityincreasing protein (BPI) in human mucosal epithelia J. Proc Natl Acad Sci USA, 2002,99(6):39023907.6Weiss J, Elsbach P, Olsson I, et al. P
29、urification and characterization of a potent bactericidal and membrane active protein from the granules of human polymorphonuclear leukocytes J. J Bio Chem,1978,253(8):26642672.7Ooi CE, Weiss J, Elsbach P, et al. A 25 kDa NHzterminal fragment carries all the antibacterial activities of the human neu
30、trophi1 60 kDa bactericidal/ permeabilityincreasing protein J. J Biol Chem, 1987, 262(31):1489114894.8Ooi CE, Weiss J, Doerfler ME, et al. Endotoxinneutralizing properties of the 25 kD Nterminal fragment and a newly isolated 30 kD Cterminal fragment of the 5560 kD bactericidal/permeabilityincreasing
31、 protein of human neutrophils J. J Exp Med, 1991,174(3):649655.9GazzanoSantoro H, Parent JB, Grinna L, et al. Highaffinity binding of the bactericidal/permeabilityincreasing protein and a recombinant aminoterminal fragment to the lipid A region of lipopolysaccharide J. Infect Immun, 1992, 60(11):475
32、44761.10Weiss J, Elsbach P, Shu C, et al. Human bactericidal/ permeabilityincreasing protein and a recombinant NH2terminal fragment cause killing of serumresistant gramnegative bacteria in whole blood and inhibit tumor necrosis factor release induced by the bacteria J. J Clin Invest, 1992, 90(3):112
33、21130.11Horwitz AH, Leigh SD, Abrahamson S, et al. Expression and characterization of cysteinemodified variants of an aminoterminal fragment of bactericidal/permeabilityincreasing protein J. Protein Expr Purif, 1996, 8(1):2840.12Schultz H, Csernok E, Johnston TW, et al. Use of native and recombinant
34、 bactericidal/permeabilityincreasing proteins (BPI) as antigens for detection of BPIANCAJ. J Immunol Methods, 1997, 205(2):127133.13Bulow E, Gullberg U, Olsson I. Structural requirements for intracellular processing and sorting of bactericidal/permeabilityincreasing protein (BPI): comparison with li
35、popolysaccharidebinding protein J. J Leukoc Biol, 2000, 68(5):669678./Porganization system and working mode, the selection of a number of municipal strengthening group to build innovation and strengthen the organization of the basic level organizations advanced model.(seven) the manual work of the C
36、ommunist Youth League Organization published and League branch of manual work, to promote the use of two manuals in the citys organizations at various levels. The county committee, directly under the group (work) is under the jurisdiction of grassroots League and group (total) using branch ratio sha
37、ll not be less than 70%.Three, to further strengthen the groups leadership and team building(a) to strengthen the leagues leadership at all levels of construction. Continue to do a good job of municipal Party Committee Organization Department on the strengthening of the Communist Youth League cadres
38、 management opinions implementation. To promote the group counties (autonomous counties, cities) Commission and city directly under the group (workers) appointed to carry out the Five team building activities. Improve the League Association Department working mechanism. Conscientiously fulfill the d
39、uties of CO, combined with the city group directly under (the) Committee of the general election and the adjustment, actively jointly with the unit where the party organization department good group leadership selection with the work. Do a good job in municipal Party committee assistant committee le
40、adership and leading cadres of the annual assessment work, adhere to and improve the assistant secretary of the Communist Youth League talk system group To weave individual conversation and collective conversation, to strengthen the committees of all levels, the Standing Committee of the Standing Co
41、mmittee, the establishment of a sound system of central group learning, the system of investigation and study, the system of important issues and the reporting system.(two) to strengthen the work of education and training of cadres to conscientiously implement the Central Committee. On the implement
42、ation of grassroots cadres training project and opinions on carrying out the mission municipal CYL Cadres training project implementation opinions, full implementation of the project of the City Youth League cadres training education. Focus on the county grassroots cadres and workers focused on trai
43、ning, the new school system vocational training League cadres, for the Municipal Organization Department jointly held the third session of the Communist Youth League system training cadres in party. Focusing on the construction of new socialist countryside, strengthening village cadres and rural con
44、struction team group member of the young team, the county organizations at various levels throughout the year is expected to train 100 00 rural grassroots cadres, plans to train 1000 village secretary of the village Party branch.(three) actively promote grassroots cadres election work. Under the uni
45、fied leadership of the party organization, to strengthen grassroots organizations direct work, standardize the election work rules and procedures, in the designated candidate, open competition to democratically elected branch secretary at the same time, actively and steadily the development of Towns
46、hip, streets and other grassroots organizations in the direct election of the pilot work, on the basis of summarizing the experience and gradually expand the scope of the pilot, and gradually formed a set of measures and cadres election system.(four) to strengthen the cadres testing exercise. To stu
47、dy the development of XX city from 2006 to 2008 cadres testing exercise planning. To select outstanding cadres to the central mission in Beijing, Shanghai, Guangzhou and other central organs and sending work in developed areas. With the Central Committee the implementation of the western region and
48、ethnic minorities cadres training plan . To carry out the eighth batch of municipal organs attachment cadre management, selection, training work. The outstanding cadres to start the National Development Bank XX branch testing exercise.(five) to strengthen the group construction. The main position to play the role of Youth League cadre education and training as th