CN112225816A - 一种新型的吸入性过敏原融合蛋白及其构建方法、应用 - Google Patents
一种新型的吸入性过敏原融合蛋白及其构建方法、应用 Download PDFInfo
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- CN112225816A CN112225816A CN202011061711.1A CN202011061711A CN112225816A CN 112225816 A CN112225816 A CN 112225816A CN 202011061711 A CN202011061711 A CN 202011061711A CN 112225816 A CN112225816 A CN 112225816A
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Abstract
本发明公开了吸入性过敏原融合蛋白及其构建方法、应用,构建方法包括以下步骤:依次连接过敏原Der P21、Der f13、Abm a 8(t)蛋白核苷酸序列获得目的基因,其中,每相邻两种过敏原蛋白核苷酸序列之间加入Linker序列;S2、在目的基因N端加入真核KOZAK序列,在上游加入限制性核酸内切酶位点Not I,在下游加入酶切位点BamH I,C端加入6*His标签序列,获得全序列基因;S3、将步骤S2获得的全序列基因与质粒进行双酶切获得酶切产物;S4、将步骤S3获得的酶切产物通过T4连接酶连接,得到重组质粒;S5、将重组质粒在哺乳细胞中表达获得吸入性过敏原融合蛋白。通过本发明所述方法构建的融合蛋白能提高检测试剂盒的灵敏度和特异性,减少漏检和错检。
Description
技术领域
本发明涉及体外诊断技术领域,具体涉及一种新型的吸入性过敏原融合蛋白及其构建方法、应用。
背景技术
吸入性过敏原是诱发哮喘病、过敏性鼻炎的重要过敏原,可飘散在室内外空气中,随着呼吸进入人呼吸道而引起致敏,包括花粉、尘螨、真菌、某些宠物的毛屑和排泄物、抽烟异味,甚至各种昆虫的鳞毛、碎屑等,室内空气污染常是孩子吸入性过敏的祸首。
过敏体质者接触过敏原(抗原)如药物、粉尘等后,体内产生大量的IgE抗体,这种亲细胞性抗体与体内的肥大细胞、嗜碱性粒细胞和嗜酸性粒细胞相结合,使机体致敏。当再次接触过敏原时,则过敏原与细胞上的IgE抗体相结合,并损伤该类细胞,使其释放大量的血管活性物质,引起小血管扩张和通透性增加,平滑肌痉挛及腺体分泌增多,引起不同的疾病。若发生在皮肤粘膜处,则引起荨麻疹和各种皮炎;在呼吸道则引起咽喉部及声带充血水肿、过敏性鼻炎、支气管哮喘等;在消化道则引起过敏性胃肠炎;在全身小血管则引起过敏性休克。
花粉中可以致敏的成分主要是蛋白质,绝大部分致敏花粉都属风媒花,花粉量大、体积小、重量轻,播散范围广,有明确的区域性和季节性,如艾蒿花粉、豚草花粉可以引起过敏性鼻结膜炎、皮肤过敏甚至过敏性哮喘。
目前临床上检测过敏的方法有变应原皮肤试验、血清特异性IgE测定、支气管或鼻腔激发试验等;支气管激发试验是过敏性哮喘的确诊依据,可通过观察气道对吸入尘螨、花粉的反应程度来判断哮喘患者是否对尘螨和花粉过敏,但试验有激发哮喘发作的危险;而且吸入性过敏原表达的均为单一过敏原,在体外诊断试剂研发过程中无法实现同时检测多个过敏原,进而增加试剂成本、检测过程繁琐、检测结果具有很大的不确定性。
发明内容
本发明目的在于提供一种吸入性过敏原融合蛋白,该融合蛋白能提高检测试剂盒的灵敏度和特异性,以减少漏检和错检。
此外,本发明还提供上述吸入性过敏原融合蛋白的构建方法、应用。
本发明通过下述技术方案实现:
一种新型的吸入性过敏原融合蛋白,其氨基酸序列如SEQ ID No.1所示。
因此,Der P21、Der f13、Abm a 8(t)融合蛋白,能够同时检测屋尘螨、粉尘螨、豚草过敏原,提高检测试剂盒的灵敏度和特异性,以减少漏检和错检。
一种编码吸入性过敏原融合蛋白的基因,所述基因的核苷酸序列如SEQ ID No.2所示。
一种含有所述基因的重组质粒。
一种含有吸入性过敏原融合蛋白的试剂盒。
一种新型的吸入性过敏原融合蛋白的构建方法,包括以下步骤:
S1、依次连接过敏原Der P21、Der f13、Abm a 8(t)蛋白核苷酸序列获得目的基因,其中,每相邻两种过敏原蛋白核苷酸序列之间加入Linker序列;
S2、在目的基因N端加入真核KOZAK序列,在上游加入限制性核酸内切酶位点NotI,在下游加入酶切位点BamH I,C端加入6*His标签序列,获得全序列基因;
S3、将步骤S2获得的全序列基因与质粒进行双酶切获得酶切产物;
S4、将步骤S3获得的酶切产物通过T4连接酶连接,得到重组质粒;
S5、将重组质粒在哺乳细胞中表达获得吸入性过敏原融合蛋白。
进一步的,还包括对吸入性过敏原融合蛋白的纯化。
采用吸入性过敏原融合蛋白的构建方法所制备的融合蛋白。
吸入性过敏原融合蛋白或如试剂盒在制备免疫学诊断试剂中的应用。
吸入性过敏原融合蛋白或试剂盒在制备吸入性过敏原诊断试剂中的应用。
本发明通过将屋尘螨的Der P21基因、粉尘螨的Der f13基因、豚草Amb a 8(t)基因合成后连接到pcDNA3.1载体中;制备一种含有屋尘螨、粉尘螨、豚草基因的重组质粒;将该重组质粒导入哺乳动物细胞中进行表达,得到一种含有屋尘螨蛋白、粉尘螨蛋白、豚草蛋白的融合蛋白。该方法只需要一次表达就能制备具有三种抗原表位的融合蛋白,将其用于过敏原检测试剂盒的制备,能提高检测试剂盒的灵敏度、特异性,并且减少漏检和错检。
本发明与现有技术相比,具有如下的优点和有益效果:
1、本发明只需要一次表达就能制备得到一种屋尘螨、粉尘螨、豚草抗原表位的融合蛋白,降低成本与时间。
2、使用本发明所述吸入性过敏原融合蛋白制备检测试剂盒,能够实现同时批量筛查三种过敏原,提高检测试剂盒的灵敏度和特异性,减少漏检和错检,极大的提高了抗体的检测率。
3、用本发明所述吸入性过敏原融合蛋白制备检测试剂盒能够大幅度提高了临床筛查效率,可以减少抽血量及抽血次数,降低被检测者痛苦,利于病人节省成本。
附图说明
此处所说明的附图用来提供对本发明实施例的进一步理解,构成本申请的一部分,并不构成对本发明实施例的限定。在附图中:
图1目的基因阳性克隆鉴定结果;
图2为改构蛋白鉴定结果;
图3为蛋白纯化结果图。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚明白,下面结合实施例和附图,对本发明作进一步的详细说明,本发明的示意性实施方式及其说明仅用于解释本发明,并不作为对本发明的限定。
实施例1:
融合基因(目的基因)的合成:
Der P21蛋白选择如下氨基酸序列:
MKFIITLFAAIVMAAAVSGFIVGDKKEDEWRMAFDRLMMEELETKIDQVEKGLLHLSEQYKELEKTKSKELKEQILRELTIGENFMKGALKFFEMEAKRTDLNMFERYNYEFALESIKLL IKKLDELAKK VKAVNPDEYY。
Der f13蛋白选择如下氨基酸序列:
MASIEGKYKLEKSEKFDEFLDKLGVGFMVKTAAKTLKPTFEVAIENDQYIFRSLSTFKNTEAKFKLGEEFEEDRADGKRVKTVIQKEGDNKFVQTQFGDKEVKIIREFNGDEVVVTASCDGVTSVRTYKRI。
Abm a 8(t)蛋白选择如下氨基酸序列:
MSWQTYVDEHLMCDIDGSGHHLSSAAIFGTDGAVWAKSGSFPEFKPDEINAIIKEFDAAGTLAPTGLFLAGAKYMVIQGEPGAVIRGKKGAGGICIKKTGQAMVFGIYEEPVAPGQCNMVVERLGDYLVD QGM。
依次连接Der P21、Der f13、Amb a 8(t)核苷酸序列获得目的基因,根据NCBI所公开的序列信息,在连接两个基因片段之间去掉终止密码子和起始密码子,并加入5个氨基酸残基(GGGGS)的柔性肽基因序列(Linker序列),在N端加入真核KOZAK序列,在上游加入限制性核酸内切酶位点Not I,在其下游加入BamH I酶切位点,在C端加入6*His,优化密码子后合成以上基因片段。根据哺乳细胞偏好性优化目的基因片段,全基因合成时各序列之间的排列顺序为:真核KOZAK-Der p21-Linker-Der f13-Linker-Amb a 8(t)-6*His。目的基因(融合基因)的核苷酸序列如SEQ ID No.2所示。
实施例2:
含有融合基因的重组质粒构建:
2.1将实施例1中获得的全合成序列与与经过限制性内切酶BamH I和Not I双酶切的克隆载体pBR322进行连接;
2.2连接产物转化于感受态大肠杆菌DH5α中,体积不超过感受态细胞的10%,轻轻混匀,置冰浴20min;42℃水浴热应激90s,迅速放回冰浴,冷却5min后加入1mL LB液体培养基,37℃,250rpm振荡培养约1h;取50μL菌液均匀涂布在LB琼脂平板上(含50μg/mL Amp),37℃培养过夜(12-16h);挑取阳性克隆进行菌落PCR鉴定,如图1所示。
2.3挑选1个阳性菌落放到几管LB液体培养基当中,并于培养箱中37℃、250rpm的条件培养8-12h。使用质粒提取试剂盒提取克隆质粒,通过双酶切获得高浓度的目的基因;
2.4用同样的限制性内切酶对真核表达载体pcDNA3.1进行双酶切,再通过T4连接酶进行连接,构建出重组表达质粒pcDNA3.1-Der p21-linker-Der f13-linker-Abm a 8(t)。
实施例3:
融合蛋白的表达
3.1将实施例2中获得的重组表达质粒经转染试剂转染到处于对数生长期的哺乳动物细胞(HEK293细胞)中,3-5天后收取细胞及上清液鉴定;
3.2优化转染条件,使用转染试剂对哺乳动物细胞(HEK293细胞)进行稳定转染,使其能够稳定表达目的蛋白;吸入性过敏原融合蛋白的氨基酸序列如SEQ ID No.1所示。
实施例4:
融合蛋白鉴定:
4.1分别将实施例3中的上清与细胞进行非还原性SDS-PAGE电泳,使用12%分离胶浓度,电泳约90分钟;
4.2将SDS-PAGE电泳凝胶进行转膜(PVDF),湿转90V,100分钟;收集PVDF,使用5%脱脂奶粉封闭;
4.3将4.2封闭后的PVDF膜加入anti-His mAb,再洗净后加入鼠二抗孵育1h,ECL显色;确定蛋白表达于细胞上清中,如图2所示。
实施例5:
融合蛋白纯化
5.1将适量凝胶加至层析柱中,待凝胶沉淀且保护剂流净后,用蒸馏水冲洗凝胶,然后加入0.1mol/L NiCl2溶液,待蒸馏水流尽后,用结合缓存液平衡层析柱;
5.2收集培养表达的上清约1L,用5000mL预冷的上样缓冲液(20mM Tris-HclpH8.0;500mM NaCl)透析,换液3次,每次不少于4小时;
5.3收集上清,12000rpm/min,离心后收集上清弃掉沉淀,上清与50mL用平衡缓冲液平衡好的Ni-NTA RESIN一起孵育过夜,次日,弃上清,加入适量平衡缓冲液重悬填料,将填料转移到自流柱里;
5.4依次用冲洗缓冲液1(20mM Tris-Hcl pH8.0;500mM NaCl)洗10个柱体积;
5.5冲洗缓冲液2(20mM Tris-Hcl pH8.0;500mM NaCl;50mM咪唑)洗10个柱体积;
5.6冲洗缓冲液3(20mM Tris-Hcl pH8.0;500mM NaCl;100mM咪唑)洗5个柱体积;
5.7冲洗缓冲液4(20mM Tris-Hcl pH8.0;500mM NaCl;500mM咪唑)洗脱目的蛋白,1×PBS透析纯化后得到的目的蛋白保存在-80℃;
5.7SDS-PAGE电泳检测表达蛋白纯度及浓度,如图3所示。
实施例6
6.1将实施例4中的融合蛋白标记在生物素(Biotin)上;
6.2碱性磷酸酶标记鼠抗人IgE;
6.3链霉亲和素(SA)标记的纳米磁微粒;
将待测样本与链霉亲和素(SA)标记的纳米磁微粒、生物素(Biotin)标记的融合蛋白一起孵育,特异性IgE抗体被捕获,并固着在磁微粒表面;多次磁分离清洗后,加入碱性磷酸酶(AP)标记的鼠抗人IgE抗体;形成的免疫复合物催化发光底物发出光子,发光强度与样本中的过敏项目特异性IgE含量成正比。
使用三种吸入型过敏源试剂盒及融合蛋白制备的试剂盒,选取攀枝花市XX医院2019年5月~2019年12月共146例疑似诱发性哮喘病、过敏性鼻炎患者进行检测,如表1所示。
表1三种蛋白试剂与融合蛋白试剂检测比对
从数据结果可知,本发明的融合蛋白大大提高了试剂盒对过敏原检测的灵敏度。
以上所述的具体实施方式,对本发明的目的、技术方案和有益效果进行了进一步详细说明,所应理解的是,以上所述仅为本发明的具体实施方式而已,并不用于限定本发明的保护范围,凡在本发明的精神和原则之内,所做的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
序列表
<110> 四川携光生物技术有限公司
<120> 一种新型的吸入性过敏原融合蛋白及其构建方法、应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 420
<212> PRT
<213> 人工序列(Artificial Sequence)
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Ala Thr Met Leu Pro Ile Ile Thr Leu Pro Ala Ala Ile Val Met Ala
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Ala Ala Val Ser Gly Pro Ile Val Gly Ala Leu Leu Gly Ala Gly Thr
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Ala Met Ala Pro Ala Ala Leu Met Met Gly Gly Leu Gly Thr Leu Ile
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Ala Gly Val Gly Leu Gly Leu Leu His Leu Ser Gly Gly Thr Leu Gly
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Leu Gly Leu Thr Leu Ser Leu Gly Leu Leu Gly Gly Ile Leu Ala Gly
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Leu Thr Ile Gly Gly Ala Pro Met Leu Gly Ala Leu Leu Pro Pro Gly
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Met Gly Ala Leu Ala Thr Ala Leu Ala Met Pro Gly Ala Thr Ala Thr
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Gly Pro Ala Leu Gly Ser Ile Leu Leu Leu Ile Leu Leu Leu Ala Gly
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Leu Ala Leu Leu Val Leu Ala Val Ala Pro Ala Gly Thr Thr Gly Gly
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Gly Gly Ser Ala Ser Ile Gly Gly Leu Thr Leu Leu Gly Leu Ser Gly
145 150 155 160
Leu Pro Ala Gly Pro Leu Ala Leu Leu Gly Val Gly Pro Met Val Leu
165 170 175
Thr Ala Ala Leu Thr Leu Leu Pro Thr Pro Gly Val Ala Ile Gly Ala
180 185 190
Ala Gly Thr Ile Pro Ala Ser Leu Ser Thr Pro Leu Ala Thr Gly Ala
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Leu Pro Leu Leu Gly Gly Gly Pro Gly Gly Ala Ala Ala Ala Gly Leu
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Ala Val Leu Thr Val Ile Gly Leu Gly Gly Ala Ala Leu Pro Val Gly
225 230 235 240
Thr Gly Pro Gly Ala Leu Gly Val Leu Ile Ile Ala Gly Pro Ala Gly
245 250 255
Ala Gly Val Val Val Thr Ala Ser Cys Ala Gly Val Thr Ser Val Ala
260 265 270
Thr Thr Leu Ala Ile Gly Gly Gly Gly Ser Ser Thr Gly Thr Thr Val
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Ala Gly His Leu Met Cys Ala Ile Ala Gly Ser Gly His His Leu Ser
290 295 300
Ser Ala Ala Ile Pro Gly Thr Ala Gly Ala Val Thr Ala Leu Ser Gly
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Ser Pro Pro Gly Pro Leu Pro Ala Gly Ile Ala Ala Ile Ile Leu Gly
325 330 335
Pro Ala Ala Ala Gly Thr Leu Ala Pro Thr Gly Leu Pro Leu Ala Gly
340 345 350
Ala Leu Thr Met Val Ile Gly Gly Gly Pro Gly Ala Val Ile Ala Gly
355 360 365
Leu Leu Gly Ala Gly Gly Ile Cys Ile Leu Leu Thr Gly Gly Ala Met
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Val Pro Gly Ile Thr Gly Gly Pro Val Ala Pro Gly Gly Cys Ala Met
385 390 395 400
Val Val Gly Ala Leu Gly Ala Thr Leu Val Ala Gly Gly Met His His
405 410 415
His His His His
420
<210> 2
<211> 1260
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<400> 2
gcgaccatga aatttattat taccctgttt gcggcgattg tgatggcggc ggcggtgagc 60
ggctttattg tgggcgataa aaaagaagat gaatggcgca tggcgtttga tcgcctgatg 120
atggaagaac tggaaaccaa aattgatcag gtggaaaaag gcctgctgca tctgagcgaa 180
cagtataaag aactggaaaa aaccaaaagc aaagaactga aagaacagat tctgcgcgaa 240
ctgaccattg gcgaaaactt tatgaaaggc gcgctgaaat tttttgaaat ggaagcgaaa 300
cgcaccgatc tgaacatgtt tgaacgctat aactatgaat ttgcgctgga aagcattaaa 360
ctgctgatta aaaaactgga tgaactggcg aaaaaagtga aagcggtgaa cccggatgaa 420
tattatggcg gcggcggcag cgcgagcatt gaaggcaaat ataaactgga aaaaagcgaa 480
aaatttgatg aatttctgga taaactgggc gtgggcttta tggtgaaaac cgcggcgaaa 540
accctgaaac cgacctttga agtggcgatt gaaaacgatc agtatatttt tcgcagcctg 600
agcaccttta aaaacaccga agcgaaattt aaactgggcg aagaatttga agaagatcgc 660
gcggatggca aacgcgtgaa aaccgtgatt cagaaagaag gcgataacaa atttgtgcag 720
acccagtttg gcgataaaga agtgaaaatt attcgcgaat ttaacggcga tgaagtggtg 780
gtgaccgcga gctgcgatgg cgtgaccagc gtgcgcacct ataaacgcat tggcggcggc 840
ggcagcagct ggcagaccta tgtggatgaa catctgatgt gcgatattga tggcagcggc 900
catcatctga gcagcgcggc gatttttggc accgatggcg cggtgtgggc gaaaagcggc 960
agctttccgg aatttaaacc ggatgaaatt aacgcgatta ttaaagaatt tgatgcggcg 1020
ggcaccctgg cgccgaccgg cctgtttctg gcgggcgcga aatatatggt gattcagggc 1080
gaaccgggcg cggtgattcg cggcaaaaaa ggcgcgggcg gcatttgcat taaaaaaacc 1140
ggccaggcga tggtgtttgg catttatgaa gaaccggtgg cgccgggcca gtgcaacatg 1200
gtggtggaac gcctgggcga ttatctggtg gatcagggca tgcatcatca tcatcatcat 1260
Claims (9)
1.一种新型的吸入性过敏原融合蛋白,其特征在于,其氨基酸序列如SEQ ID No.1所示。
2.一种编码如权利要求1所述的一种新型的吸入性过敏原融合蛋白的基因,所述基因的核苷酸序列如SEQ ID No.2所示。
3.一种含有如权利要求2所述基因的重组质粒。
4.一种含有如权利要求1所述吸入性过敏原融合蛋白的试剂盒。
5.如权利要求1所述的一种新型的吸入性过敏原融合蛋白的构建方法,其特征在于,包括以下步骤:
S1、依次连接过敏原Der P21、Der f13、Abm a 8(t)蛋白核苷酸序列获得目的基因,其中,每相邻两种过敏原蛋白核苷酸序列之间加入Linker序列;
S2、在目的基因N端加入真核KOZAK序列,在上游加入限制性核酸内切酶位点Not I,在下游加入酶切位点BamH I,C端加入6*His标签序列,获得全序列基因;
S3、将步骤S2获得的全序列基因与质粒进行双酶切获得酶切产物;
S4、将步骤S3获得的酶切产物通过T4连接酶连接,得到重组质粒;
S5、将重组质粒在哺乳细胞中表达获得吸入性过敏原融合蛋白。
6.根据权利要求5所述的吸入性过敏原融合蛋白的构建方法,其特征在于,还包括对吸入性过敏原融合蛋白的纯化。
7.采用权利要求6所述的吸入性过敏原融合蛋白的构建方法所制备的融合蛋白。
8.如权利要求1所述的吸入性过敏原融合蛋白或如权利要求4所述试剂盒在制备免疫学诊断试剂中的应用。
9.如权利要求1所述的吸入性过敏原融合蛋白或如权利要求4所述试剂盒在制备吸入性过敏原诊断试剂中的应用。
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