CN114957487B - 一种重组结核分枝杆菌融合蛋白及其在结核诊断中应用 - Google Patents
一种重组结核分枝杆菌融合蛋白及其在结核诊断中应用 Download PDFInfo
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Abstract
本发明公开一种重组结核分枝杆菌融合蛋白及其在结核诊断中应用。本发明所述的重组结核分枝杆菌融合蛋白用于结核病的体内诊断时,该蛋白具有较高敏感性、特异性和安全性。该融合蛋白还可以用于基于γ干扰素释放实验的体外诊断试剂盒。
Description
技术领域
本发明涉及生物技术领域,具体涉及一种重组结核分枝杆菌融合蛋白及其在结核诊断中应用。
背景技术
结核病是慢性传染性疾病。目前全球已有20亿人感染结核菌,活动性结核患者达1500万,每年新发结核患者达800万~1000万,有180万人因结核病死亡。
传统的结核诊断手段包括影像学、细菌学、临床表现、分子生物技术等许多方面,其中经典的结核菌素试验已经应用了100年,但是该方法特异性差,不能鉴别卡介苗接种者,血清结核抗体检测等方法的敏感性和特异性不理想;近几年兴起的γ干扰素释放试验的试剂昂贵,技术复杂,需要特殊的设备和培训,不适合基层医疗单位或者现场应用。TB-PPD目前已经成为临床上最常用且最简便的一种结核菌感染诊断方法,反应越强,表示结核感染可能性越大。但是TB-PPD 不能区分卡介苗(BCG)接种或是结核杆菌感染,假阳性的结果影响结核病筛查。因此,临床急需一种适合基层医疗单位的皮肤诊断试剂。来源于致病性结核杆菌的ESAT-6和CFP-10两个抗原,在所有的BCG疫苗株中缺失,使得ESAT-6和 CFP-10有望成为新一代诊断试剂的核心抗原,人群中特异性可以达到93%,远高于TB-PPD的7%[1]。可用于结核菌潜伏感染人群的筛查以及结核病的辅助诊断,克服了通用的特异性γ-IFN检测方法价格昂贵、设备及操作人员要求高的缺点,使用方便、易于推广、适合于大规模普及。但是目前的重组结核ESAT-6和 CFP-10分枝杆菌融合蛋白特异性和敏感性均有待提高。
发明内容
本发明涉及一种重组结核分枝杆菌融合蛋白及其在结核诊断中应用,本发明所述的重组结核分枝杆菌融合蛋白用于结核病的体内诊断时,该蛋白具有较高敏感性、特异性和安全性。该融合蛋白还可以用于基于γ干扰素释放实验的体外诊断试剂盒。
本发明所述的重组结核分枝杆菌融合蛋白的氨基酸序列如SEQ ID NO.1所示。
本发明还提供编码所述重组结核分枝杆菌融合蛋白的核苷酸序列。所述的核酸序列可以按照本领域常规的方法进行设计,在一些实施例中,所述核苷酸序列如SEQ ID NO.2所示。
本发明所述的融合蛋白可以人工合成,也可以先合成其编码基因,再进行生物表达得到。
在一种具体的实施方式中,本发明还提供所述重组结核分枝杆菌融合蛋白的一种具体的获得方法:将本发明所述的重组融合蛋白的编码基因序列插入原核表达载体,转入菌株扩增和纯化。
本发明还提供一种重组结核分枝杆菌融合蛋白的纯化方法,扩增和纯化后的菌体采用超声或高压均质机破碎菌体后,pH=7.8~8.2的Tris-HCl作为蛋白溶液体系进行超滤;超滤结束后进行阴离子层析,5%~10%的NaCl去除杂蛋白, 10%~15%的NaCl收集目的蛋白。
本发明还提供一种结核体内诊断试剂,所述诊断试剂中含有本发明所述的重组结核分枝杆菌融合蛋白。
本发明还进一步提供一种基于γ干扰素释放实验的结核体外诊断试剂盒,所述体外诊断试剂盒中含有本发明所述的重组结核分枝杆菌融合蛋白。
本发明所述的重组结核分枝杆菌融合蛋白相对于现有技术,具有如下优势:
①特异性和敏感性相对于现有融合蛋白有显著提高。
②安全性显著提高。
③经过工艺优化之后,本发明所提供的融合蛋白纯化方法仅包含菌体破碎、超滤和离子层析3个步骤,简化了生产工艺,更有利于工业化生产。
附图说明
图1重组蛋白电泳图谱。
具体实施方式
以下结合具体实施例,对本发明进行进一步说明,若无特别说明,实施例中用到的试剂均为本领域的常规试剂,所用的方法均为本领域的常规方法。
以下实施例中对照的EC的序列如SEQ ID NO.3所示,EEC的序列如SEQ ID NO.4所示。
实施例1:SEQ ID NO.1所述的重组蛋白表达质粒与工程菌的构建
根据SEQ ID NO.1所述的氨基酸序列,根据密码子优化设计后的基因序列 (SEQID NO.2)交由第三方基因合成公司进行合成,将合成的基因插入原核表达载体。表达载体构建成功后,先将该载体转入大肠杆菌DH5α菌株内,进行质粒扩增,扩增后用纯化试剂提取质粒。最后将提取的质粒转化蛋白表达菌株(如BL-21(DE3))。将转化成功后的第一代菌种作为原始种子,分别建立主种子批和工作种子批,用于重组蛋白的表达制备。
实施例2:SEQ ID NO.1所述的重组蛋白的制备
1.工程菌发酵及重组蛋白表达:将菌种按1%接种至250mL LB培养基内, 37℃摇床培养至OD600=0.6~0.8时,加IPTG(终浓度为0.4mmol/L)诱导,继续培养4h。离心收集菌体,用0.01M PBS缓冲液洗涤两次,将洗涤后的菌体放置于4℃冰箱备用。
2.菌体破碎:用高压均质机破碎菌体,破碎压力为800bar,破碎两次,10000 ×g离心收上清。
3.超滤:将蛋白溶液体系更换为Tris-HCl(pH=8.0);超滤结束后进行阴离子层析,9%NaCl去除杂蛋白,14%的NaCl收集目的蛋白。纯化后用SDS-PAGE 电泳法检测,目的条带分子量为25.7kD,与预测一致;蛋白纯度大于95%(图1 泳道4。泳道1为Mark,泳道2为5%去杂纯化,泳道3为7%去杂纯化,泳道4 为9%去杂纯化),本发明所述的纯化方法能得到目的蛋白且纯度较高。
实施例3:实施例2制备的融合蛋白与其他结核诊断抗原药效对比
将实施例2制备的重组融合蛋白与TB-PPD、EC和EEC皮试效果进行对比。
实验设计如下:用结核分枝杆菌减毒株(H37Ra)将豚鼠致敏,致敏剂量为 1mg。三周后,将豚鼠背部脊柱两侧去毛,用酒精棉消毒去毛部位的皮肤,以轮圈法分别用1mL一次性注射器于皮内注射抗原各0.1mL。其中,重组蛋白剂量为0.5μg/只,TB-PPD 5IU/只。分别于24h和48h测量红晕或硬结大小(纵横直径相加除2)。结果如表1所示。数据表明,实施例2制备的重组融合蛋白皮试反应效果优于TB-PPD、EC和EEC。
表1.不同抗原动物皮试药效对比(mm)
实施例4:实施例2制备的融合蛋白与其他抗原体外诊断应用效果对比对比
分别将实施例2制备的融合蛋白、EC和EEC作为γ干扰素释放试验的刺激剂,比较三种抗原的刺激效果。具体实施步骤如下:
1.阳性血采集:每人采集静脉血10mL,共采集42份;
2.抗原稀释:将3种重组结核杆菌抗原稀释至5、10、20μg/mL,(稀释液为含0.0005%吐温80的0.01M PBS缓冲液(pH7.2~7.4));
3.样本采集分装:新鲜采集的全血样本混匀后按1mL/管分装;
4.刺激剂添加:采集样本后6小时内完成刺激剂添加,每管全血样本加入 50μL抗原;
5.孵育:在恒温培养箱内37℃下孵育18-24小时;
6.ELISA检测:将刺激后的全血离心收集血浆,取50μL加入抗γ干扰素抗体包被的96孔板中,37℃下孵育1h后再依次加入二抗、酶、显色剂和终止液,测定OD并判定结果。
结果如表2所示,在3种剂量条件下,实施例2制备的融合蛋白作为刺激剂获得的阳性率显著高于EC和EEC。
表2不同抗原作为γ干扰素释放试验刺激剂检出阳性率对比
实施例5:实施例2制备的融合蛋白与EEC致敏效应对比
试验组和对照组分别选用体重300~400g未做过任何试验的豚鼠各3只,试验组每只豚鼠皮内注射10μg、20μg、40μg、80μg、100μg实施例2制备的融合蛋白或EEC,共3次,每次间隔5天。在第3次注射后15天,试验组与对照组每只豚鼠各皮内注射相应剂量的抗原,连续观察3天,记录两组动物反应。结果如表3可见,EEC在60μg剂量时在接种部位产生了明显红晕,而100μg剂量时,除产生红晕外,接种部位皮肤有坏死现象;实施例2制备的融合蛋白在剂量达到100μg时才产生红晕。因此,本申请实施例2制备的融合蛋白的致敏效应显著降低,安全性增加。
表3.实施例2制备的融合蛋白与EEC致敏效应结果
序列表
<110> 北京祥瑞生物制品股份有限公司
<120> 一种重组结核分枝杆菌融合蛋白及其在结核诊断中应用
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 245
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Met Gln Gln Trp Asn Phe Ala Gly Ile Glu Ala Ala Ala Ser Ala Ile
1 5 10 15
Gln Gly Asn Val Thr Ser Ile His Ser Leu Leu Asp Glu Gly Lys Gln
20 25 30
Ser Leu Thr Lys Leu Ala Ala Ala Trp Gly Gly Ser Gly Ser Glu Ala
35 40 45
Tyr Gln Gly Val Gln Gln Lys Trp Asp Ala Thr Ala Thr Glu Leu Asn
50 55 60
Asn Ala Leu Gln Asn Leu Ala Arg Thr Ile Ser Glu Ala Gly Gln Ala
65 70 75 80
Met Ala Ser Gln Gln Trp Asn Phe Ala Gly Ile Glu Ala Ala Ala Ser
85 90 95
Ala Ile Gln Gly Asn Val Thr Ser Ile His Ser Leu Leu Asp Glu Gly
100 105 110
Lys Gln Ser Leu Thr Lys Leu Ala Ala Ala Trp Gly Gly Ser Gly Ser
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Glu Ala Tyr Gln Gly Val Gln Gln Lys Trp Asp Ala Thr Ala Thr Glu
130 135 140
Leu Asn Asn Ala Leu Gln Asn Leu Ala Arg Thr Ile Ser Glu Ala Gly
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Gln Ala Met Ala Ser Thr Asp Ala Ala Thr Leu Ala Gln Glu Ala Gly
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Asn Phe Glu Arg Ile Ser Gly Asp Leu Lys Thr Gln Ile Asp Gln Val
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Glu Ser Thr Ala Gly Ser Leu Gln Gly Gln Trp Arg Gly Ala Ala Gly
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Thr Ala Ala Gln Ala Ala Val Val Arg Phe Gln Glu Ala Ala Asn Lys
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Gln Lys Gln Glu Leu Asp Glu Ile Ser Thr Asn Ile Arg Gln Ala Gly
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Val Gln Tyr Ser Arg
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<210> 2
<211> 735
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atgcagcagt ggaatttcgc tggtattgaa gcggcagcat ccgctatcca gggtaacgtg 60
accagcatcc atagcctgct ggacgaaggc aaacagagcc tgactaaact ggcagcagcg 120
tggggcggtt ctggttctga agcgtatcag ggtgtccagc agaagtggga tgcaaccgca 180
accgagctga acaacgccct gcagaacctg gcgcgtacca tctctgaagc tggtcaggcg 240
atggcgtctc agcagtggaa ctttgcaggt atcgaagcgg cggcatctgc gatccagggc 300
aacgttacct ctatccactc cctgctggac gaaggcaagc agtccctgac taaactggct 360
gcagcctggg gcggttctgg ttctgaagct taccagggtg ttcagcagaa atgggacgcg 420
actgcgaccg aactgaataa cgcactgcag aacctggctc gcaccatctc tgaagcgggt 480
caggctatgg cttccactga tgctgcaacc ctggcgcagg aagcaggcaa cttcgaacgt 540
atctctggtg acctgaaaac ccagattgac caggtcgaat ctaccgcagg ttctctgcaa 600
ggccaatggc gtggtgctgc aggtaccgct gctcaggctg ctgtagtacg tttccaagaa 660
gcggcgaaca agcaaaaaca ggaactggac gaaatttcca ccaacatccg tcaagcgggc 720
gtccagtatt ctcgc 735
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<211> 195
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Met Thr Glu Gln Gln Trp Asn Phe Ala Gly Ile Glu Ala Ala Ala Ser
1 5 10 15
Ala Ile Gln Gly Asn Val Thr Ser Ile His Ser Leu Leu Asp Glu Gly
20 25 30
Lys Gln Ser Leu Thr Lys Leu Ala Ala Ala Trp Gly Gly Ser Gly Ser
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Glu Ala Tyr Gln Gly Val Gln Gln Lys Trp Asp Ala Thr Ala Thr Glu
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Leu Asn Asn Ala Leu Gln Asn Leu Ala Arg Thr Ile Ser Glu Ala Gly
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Gln Ala Met Ala Ser Thr Glu Gly Asn Val Thr Gly Met Phe Ala Met
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Ala Glu Met Lys Thr Asp Ala Ala Thr Leu Ala Gln Glu Ala Gly Asn
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Gln Tyr Ser Arg Ala Asp Glu Glu Gln Gln Gln Ala Leu Ser Ser Gln
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<210> 4
<211> 290
<212> PRT
<213> 人工序列(Artificial Sequence)
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Met Thr Glu Gln Gln Trp Asn Phe Ala Gly Ile Glu Ala Ala Ala Ser
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Ala Ile Gln Gly Asn Val Thr Ser Ile His Ser Leu Leu Asp Glu Gly
20 25 30
Lys Gln Ser Leu Thr Lys Leu Ala Ala Ala Trp Gly Gly Ser Gly Ser
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Glu Ala Tyr Gln Gly Val Gln Gln Lys Trp Asp Ala Thr Ala Thr Glu
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Leu Asn Asn Ala Leu Gln Asn Leu Ala Arg Thr Ile Ser Glu Ala Gly
65 70 75 80
Gln Ala Met Ala Ser Thr Glu Gly Asn Val Thr Gly Met Phe Ala Met
85 90 95
Thr Glu Gln Gln Trp Asn Phe Ala Gly Ile Glu Ala Ala Ala Ser Ala
100 105 110
Ile Gln Gly Asn Val Thr Ser Ile His Ser Leu Leu Asp Glu Gly Lys
115 120 125
Gln Ser Leu Thr Lys Leu Ala Ala Ala Trp Gly Gly Ser Gly Ser Glu
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Ala Tyr Gln Gly Val Gln Gln Lys Trp Asp Ala Thr Ala Thr Glu Leu
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Asn Asn Ala Leu Gln Asn Leu Ala Arg Thr Ile Ser Glu Ala Gly Gln
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Ala Met Ala Ser Thr Glu Gly Asn Val Thr Gly Met Phe Ala Met Ala
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Glu Met Lys Thr Asp Ala Ala Thr Leu Ala Gln Glu Ala Gly Asn Phe
195 200 205
Glu Arg Ile Ser Gly Asp Leu Lys Thr Gln Ile Asp Gln Val Glu Ser
210 215 220
Thr Ala Gly Ser Leu Gln Gly Gln Trp Arg Gly Ala Ala Gly Thr Ala
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Ala Gln Ala Ala Val Val Arg Phe Gln Glu Ala Ala Asn Lys Gln Lys
245 250 255
Gln Glu Leu Asp Glu Ile Ser Thr Asn Ile Arg Gln Ala Gly Val Gln
260 265 270
Tyr Ser Arg Ala Asp Glu Glu Gln Gln Gln Ala Leu Ser Ser Gln Met
275 280 285
Gly Phe
290
Claims (10)
1.重组结核分枝杆菌融合蛋白,其特征在于,氨基酸序列如SEQ ID NO.1所示。
2.编码权利要求1所述的重组结核分枝杆菌融合蛋白的基因。
3.根据权利要求2所述的基因,其特征在于,核苷酸序列如SEQ ID NO.2所示。
4.包含权利要求2或3所述的基因的重组表达载体。
5.包含权利要求2或3所述的基因的菌株。
6.权利要求1所述的重组结核分枝杆菌融合蛋白的制备方法,其特征在于,将权利要求2或3所述的基因插入原核表达载体,转入菌株扩增和纯化。
7.根据权利要求6所述的方法,其特征在于,扩增和纯化后的菌体采用超声或高压均质机破碎菌体后,pH为7.8~8.2的Tris-HCl作为蛋白溶液体系进行超滤;超滤结束后进行阴离子层析,5%~10%的NaCl去除杂蛋白,10%~15%的NaCl收集目的蛋白。
8.权利要求1所述的融合蛋白、权利要求2或3所述的基因、权利要求4所述的重组表达载体或权利要求5所述的菌株在制备结核诊断试剂或检测试剂盒中的应用。
9.一种结核体内诊断试剂,其特征在于,所述诊断试剂中含有权利要求1所述的融合蛋白。
10.一种基于γ干扰素释放实验的结核体外诊断试剂盒,其特征在于,所述体外诊断试剂盒中含有权利要求1所述的融合蛋白。
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