CN114350708B - 融合表达蛋白及其制备方法和用途 - Google Patents
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- CN114350708B CN114350708B CN202210050259.1A CN202210050259A CN114350708B CN 114350708 B CN114350708 B CN 114350708B CN 202210050259 A CN202210050259 A CN 202210050259A CN 114350708 B CN114350708 B CN 114350708B
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Abstract
本发明属于生物工程技术领域,具体涉及一种融合表达蛋白及其制备方法和用途。现有炎症诊断和检测时采用的炎症项目质控品要么为单项目质控品,操作不便,要么复合质控品制备方法复杂,成本高,针对此,本发明提供了一种融合表达蛋白,开创性的将RP、SAA、PCT、IL6这4种炎症标志蛋白融合表达,得到的融合蛋白可以按照比例分别配置PCT和IL6项目复合质控及CRP和SAA项目复合质控,不仅具有较好的稳定性和准确性,同时能够有效降低质控品的成本,提高体外诊断检测的便捷性并能很好地控制质控品的批间差。
Description
技术领域
本发明属于生物工程技术领域,具体涉及一种融合表达蛋白及其制备方法和用途。
背景技术
感染性疾病是临床的常见病,多发病,其主要由细菌、病毒及真菌入侵体内引起的炎性反应,多数伴有发热情况。细菌、病毒及真菌等病原体及其产物所引起的感染性疾病,迄今仍然是人类死亡和致残的主要原因之一,威胁着人类的身体健康。因此,炎症的诊断及检测显得尤为重要。
在炎症诊断和检测中,选择合适的炎症反应标志物尤为重要。
随着Assicot等人于1993年首次报道脓毒血症患者降钙素原(PCT)水平明显升高以来,大量关于降钙素原与感染性疾病的研究相继展开。目前,PCT越来越被国际医疗界公认为是具有重要临床价值的炎症反应标志物,现已广泛应用于临床感染性疾病的诊疗,国内外医院已普遍开展检测,并将其作为一项用于细菌感染早期诊断、鉴别诊断和疗效监测的诊断指标。PCT是一种无激素活性的降钙素(CT)前肽物质,分子量为13kDa,半衰期为20-24h,该基因由2800个碱基对组成,PCT前体在内源多肽酶的作用下剪掉nPro-CT端单一序列,生成116个氨基酸的PCT。在健康人血浆中PCT的含量极少(<0.0025ug/L),且体内稳定性好,体内血清PCT浓度额改变基于感染的严重程度和感染额类型。
C反应蛋白(CRP)是1930年由Tmet和Francis首次在急性大叶肺炎患者的血清中发现一种能与肺炎球菌细胞壁的C多糖发生特异性沉淀反应的物质,是机体在应激状态下由肝脏合成的一种急性期反应蛋白,分子量为115-140kDa,正常人血清中含量极低,当有急性炎症、创伤和冠心病时CRP会升高。
血清淀粉样蛋白A(SAA)是由104个氨基酸组成的多肽,其在天然状态时的相对分子质量为12-14kDa,血清SAA基因位于第11号染色体上。SAA是近年发现的一种敏感的炎症标志物,是一种急性反应蛋白,在急、慢性炎症反应时,SAA浓度明显升高,可达正常额1000倍以上。同时SAA在细菌和病毒感染的早期均明显升高,创伤和烧伤等应激状态下也会快速增高。
白细胞介素-6(Interleukin-6,IL-6)是白细胞介素的一种,其位于人类第7号染色体上,分子量在21-30kDa左右。据报道IL-6参与许多疾病的发生和发展,其血液水平与炎症、病毒感染、自身免疫疾病密切相关,与肿瘤、内外伤、外科手术、应激反应、脑死亡等也具有极大的相关性。IL-6的检测适用于急性炎症的早期辅助诊断。它的变化比CRP灵敏,而且持续时间长。IL-6可用于评估全身炎症反应综合征、脓血症和脓毒性休克的严重程度,并可预测相关患者的预后。IL-6也可用作检测新生儿败血症的早期预警标志物。
目前,炎症诊断和检测时采用的炎症项目质控品均为单项目质控品,操作繁琐,成本高,并且不能很好地控制批间差。
专利CN110133282A公开了一种炎症复合质控的制备方法,该专利中的复合质控品,以血清为基质,包含保护物质和炎症类标志物分析成分(CRP、SAA、PCT、IL-6、ASO中的至少两种),保护物质由赋形剂、抗氧化剂、表面活性剂、稳定剂和防腐剂等组成。该复合质控品中由于加入了保护物质,使得质控品中各分析成分稳定,同时该复合质控品包含的成分多,且稳定性良好,既方便临床操作又节约成本。但该专利的复合质控品包含的成分众多,成本显著增高,也难以工业化应用。
发明内容
本发明要解决的技术问题为:现有炎症诊断和检测时采用的炎症项目质控品要么为单项目质控品,操作不便,要么复合质控品制备方法复杂,成本高的问题。
本发明解决上述技术问题的技术方案为:提供一种表达单元,该表达单元从5’到3’依次为:Kozak基因-CRP基因-linker-SAA基因-linker-PCT基因-linker-IL6基因-组氨酸标签。
所述的Kozak基因的核苷酸序列如SEQ ID NO:6所示。
SEQ ID NO:6 Kozak基因的核苷酸序列
GCCACCATGG。
其中,所述的CRP基因的核苷酸序列如SEQ ID NO:1所示。
SEQ ID NO:1 CRP基因的核苷酸序列
ATGTCGAGGAAGGCTTTTGTGTTTCCCAAAGAGTCGGATACTTCCTATGTATCCCTCAAAGCACCGTTAACGAAGCCTCTCAAAGCCTTCACTGTGTGCCTCCACTTCTACACGGAACTGTCCTCGACCCGTGGGTACAGTATTTTCTCGTATGCCACCAAGAGACAAGACAATGAGATTCTCATATTTTGGTCTAAGGATATAGGATACAGTTTTACAGTGGGTGGGTCTGAAATATTATTCGAGGTTCCTGAAGTCACAGTAGCTCCAGTACACATTTGTACAAGCTGGGAGTCCGCCTCAGGGATCGTGGAGTTCTGGGTAGATGGGAAGCCCAGGGTGAGGAAGAGTCTGAAGAAGGGATACACTGTGGGGGCAGAAGCAAGCATCATCTTGGGGCAGGAGCAGGATTCCTTCGGTGGGAACTTTGAAGGAAGCCAGTCCCTGGTGGGAGACATTGGAAATGTGAACATGTGGGACTTTGTGCTGTCACCAGATGAGATTAACACCATCTATCTTGGCGGGCCCTTCAGTCCTAATGTCCTGAACTGGCGGGCACTGAAGTATGAAGTGCAAGGCGAAGTGTTCACCAAACCCCAGCTGTGGCCCTGA。
所述的SAA基因的核苷酸序列如SEQ ID NO:2所示。
SEQ ID NO:2 SAA基因的核苷酸序列
ATGAAGCTTCTCACGGGCCTGGTTTTCTGCTCCTTGGTCCTGGGTGTCAGCAGCCGAAGCTTCTTTTCGTTCCTTGGCGAGGCTTTTGATGGGGCTCGGGACATGTGGAGAGCCTACTCTGACATGAGAGAAGCCAATTACATCGGCTCAGACAAATACTTCCATGCTCGGGGGAACTATGATGCTGCCAAAAGGGGACCTGGGGGTGCCTGGGCTGCAGAAGTGATCACCGATGCCAGAGAGAATATCCAGAGATTCTTTGGCCATGGTGCGGAGGACTCGCTGGCTGATCAGGCTGCCAATGAATGGGGCAGGAGTGGCAAAGACCCCAATCACTTCCGACCTGCTGGCCTGCCTGAGAAATACTGA。
所述的PCT基因的核苷酸序列如SEQ ID NO:3所示。
SEQ ID NO:3 PCT基因的核苷酸序列
ATGGGCTTCCAAAAGTTCTCCCCCTTCCTGGCTCTCAGCATCTTGGTCCTGTTGCAGGCAGGCAGCCTCCATGCAGCACCATTCAGGTCTGCCCTGGAGAGCAGCCCAGCAGACCCGGCCACGCTCAGTGAGGACGAAGCGCGCCTCCTGCTGGCTGCACTGGTGCAGGACTATGTGCAGATGAAGGCCAGTGAGCTGGAGCAGGAGCAAGAGAGAGAGGGCTCCAGCCTGGACAGCCCCAGATCTAAGCGGTGCGGTAATCTGAGTACTTGCATGCTGGGCACATACACGCAGGACTTCAACAAGTTTCACACGTTCCCCCAAACTGCAATTGGGGTTGGAGCACCTGGAAAGAAAAGGGATATGTCCAGCGACTTGGAGAGAGACCATCGCCCTCATGTTAGCATGCCCCAGAATGCCAACTAA。
所述的IL6基因的核苷酸序列如SEQ ID NO:4所示。
SEQ ID NO:4 IL6基因的核苷酸序列
ATGAACTCCTTCTCCACAAGCGCCTTCGGTCCAGTTGCCTTCTCCCTGGGGCTGCTCCTGGTGTTGCCTGCTGCCTTCCCTGCCCCAGTACCCCCAGGAGAAGATTCCAAAGATGTAGCCGCCCCACACAGACAGCCACTCACCTCTTCAGAACGAATTGACAAACAAATTCGGTACATCCTCGACGGCATCTCAGCCCTGAGAAAGGAGACATGTAACAAGAGTAACATGTGTGAAAGCAGCAAAGAGGCACTGGCAGAAAACAACCTGAACCTTCCAAAGATGGCTGAAAAAGATGGATGCTTCCAATCTGGATTCAATGAGGAGACTTGCCTGGTGAAAATCATCACTGGTCTTTTGGAGTTTGAGGTATACCTAGAGTACCTCCAGAACAGATTTGAGAGTAGTGAGGAACAAGCCAGAGCTGTGCAGATGAGTACAAAAGTCCTGATCCAGTTCCTGCAGAAAAAGGCAAAGAATCTAGATGCAATAACCACCCCTGACCCAACCACAAATGCCAGCCTGCTGACGAAGCTGCAGGCACAGAACCAGTGGCTGCAGGACATGACAACTCATCTCATTCTGCGCAGCTTTAAGGAGTTCCTGCAGTCCAGCCTGAGGGCTCTTCGGCAAATGTAG。
所述的linker的序列如下所示。
EAAAKEAAAKEAAAK。
本发明还提供了一种包含上述表达单元的重组载体。
不优选的,所述重组载体中,表达载体为pcDNA3.1。
本发明还提供了一种包含上述表达单元或重组载体的宿主细胞。
所述的宿主细胞为人HEK 293T细胞。
本发明还提供了一种由上述表达单元、重组载体或宿主细胞表达得到的融合蛋白。
本发明还提供了一种上述融合蛋白的制备方法,包括以下步骤:
a、筛选包含RP、SAA、PCT和IL6的目的基因,构建重组载体;
b、将步骤a得到的重组载体导入人HEK 293T细胞,经表达纯化后,得到融合蛋白。
上述融合蛋白的具体制备方法为,包括以下步骤:
a、依次合成获得CRP、SAA、PCT和IL6基因完整的CDS区,在基因之间插入刚性linker;在CRP基因起始密码子ATG前插入Hind III限制性内切酶酶切位点和Kozak序列,在IL6基因下游插入6*His标签及Xba I酶切位点;
b、将步骤a得到的目的基因与哺乳动物表达载体pcDNA3.1质粒通过Hind III和Xba I双酶切,用胶回收试剂盒纯化后用T4连接酶连接,经鉴定后获得重组载体;
c、将获得的重组载体瞬时转染至人HEK 293T,使其在细胞中转录翻译并得到同时表达CRP、SAA、PCT和IL6基因的融合蛋白质,72h后收集裂解细胞;
d、利用插入的His标签通过Ni-NTA纯化得到融合蛋白。
本发明还提供了一种上述融合蛋白的用途,作为复合质控品用于制备炎症项目检测试剂盒。
本发明首先开创性的将4种炎症标志蛋白融合表达,同时为了防止4个不同基因编码蛋白质后结构域之间交叠发生相互作用影响蛋白质最终的功能,在每两个基因序列之间插入刚性Linker EAAAKn(n=3),能够控制最佳距离,有效的将两端的蛋白质结构域分开,保证Linker两端蛋白质相对独立的结构及生物学功能。接着将其利用哺乳动物表达载体在人源细胞系中成功表达,得到与人体内蛋白质相同结构与功能的融合蛋白,提高蛋白质的活性。进一步通过设计的6*His标签对表达的融合蛋白进行纯化得到纯度单一的蛋白质。将其作为原料可以按照比例分别配置PCT和IL6项目复合质控及CRP和SAA项目复合质控,不仅具有较好的稳定性和准确性,同时能够有效降低质控品的成本,提高体外诊断检测的便捷性并能很好地控制质控品的批间差。本发明的融合蛋白作为复合质控品,效价高,特异性好,稳定性高,成本低,可以提高体外诊断行业检测便捷性。
附图说明
图1为CRP、SAA、PCT和IL6融合蛋白设计示意图。
图2为重组载体鉴定结果图;M:DNAmarker;1:酶切重组载体。
图3为重组蛋白鉴定结果图;对照:HEK293T细胞;超表达重组载体:转染重组载体的HEK293T细胞。
图4为蛋白纯化结果图;1:未纯化蛋白质;2:已纯化蛋白质;M:蛋白Marker。
具体实施方式
本发明的关键点在于将炎症CRP、SAA、PCT、IL6四项目相关基因通过特定刚性Linker EAAAKn(n=3)连接后融合表达,从而作为体外诊断试剂盒中炎症PCT和IL6项目复合质控及CRP和SAA项目复合质控。
为使本发明的目的、技术方案和优点更加清楚明白,下面结合实施案例和附图,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合成目的基因
在NCBI中查询4个基因完整CDS区,其中,CRP基因、SAA基因、PCT基因、IL6序列的核苷酸序列分别如SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4所示。
在CRP起始密码子ATG前插入Hind III限制性内切酶酶切位点和Kozak序列,其中Hind III限制性内切酶酶切位点的核苷酸序列如SEQ ID NO:5所示(AAGCTT),Kozak的核苷酸序列如SEQ ID NO:6所示(GCCACCATGG),本发明中插入Kozak序列的目的是为了保证目的基因转录产物能够被有效的翻译。
在IL6终止密码子TAG前插入6*His标签,核苷酸序列如SEQ ID NO:7所示(CATCATCATCATCATCAC),在TAG后插入Xba I酶切位点,核苷酸序列如SEQ ID NO:8所示(TCTAGA)。同时,删除CRP、SAA、PCT的终止密码子,并在基因与基因之间插入刚性linker,序列如下所示(EAAAKEAAAKEAAAK),使得4个编码不同基因的蛋白质结构域之间不会发生交叠并保持他们独立的功能。
实施例中全基因合成的示意图见图1。
Kozak—CRP—linker—SAA—linker—PCT—linker—IL6—6*His。
实施例2重组载体的构建
将实施例1合成获得的目的基因与哺乳动物表达载体pcDNA3.1同时用Hind III和Xba I限制性内切酶消化30min,酶切体系见下表1所示。酶切产物经过琼脂糖凝胶电泳检测后回收备用。
表1酶切体系
试剂 | 体积/μL |
pcDNA3.1/重组目的基因 | 3.6/2.6 |
10×Cut buffer | 4 |
Hind III | 1.5 |
Xba I | 1.5 |
无菌水 | 补齐至40μL |
将回收所得目的基因片段以6倍载体质量比与pcDNA3.1用T4 DNA连接酶16℃过夜连接,连接后的产物转化至Top 10感受态细胞中,涂板于含50μg/mL氨苄青霉素的LB固体培养基,在37℃恒温培养箱静置倒置培养12-16小时。
在超净工作台中分别挑取10个阳性单克隆进行菌落PCR验证,将菌落PCR验证正确的重组载体送至测序公司鉴定获得成功重组载体。将重组载体进一步转化酶切鉴定,酶切大小约为5400bp和2100bp左右,结果见图2所示。结果表明成功获得重组载体。
实施例3融合蛋白的表达及鉴定
(1)将实施例2成功获得的重组载体进一步培养后用无内毒素质粒提取试剂盒获得大量重组质粒备用。
(2)复苏HEK 293T细胞,经过细胞传代后培养细胞至其生长至70-80%左右密度时即可转染。
(3)Opti-MEM稀释质粒和Lipofectamine 2000转染试剂后静置5min,混合质粒及转染试剂后静置30min,均匀加入细胞培养皿,于细胞培养箱培养。
(4)细胞培养至72h后,从培养箱取出细胞,PBS清洗残留培养基,用RIPA裂解液(含蛋白酶抑制剂和PMSF)裂解细胞,用黄枪头粗的部分刮细胞,然后吸出液体加入到离心管中,冰上静置30min后4℃,12000rpm离心10min,转移上清液至新的离心管中,加入终浓度为1×的SDS-聚丙烯酰胺凝胶(SDS-PAGE)上样缓冲液,沸水煮10min后于-20℃保存备用。
(5)配制分离胶和浓缩胶。点样时每个胶孔点样约20μg蛋白,80V电压电泳1h后调整电压至120V电泳2h。用湿转法将目的蛋白转移至0.45μm PVDF膜上,湿转结束后用5%脱脂奶粉封闭。分别加入一抗工作液,4℃过夜孵育,并以GAPDH作为内参蛋白。加入HRP标记的二抗,37℃孵育2h。使用ECL发光液显色,使用Gene Gnome XRQ化学发光系统收集信号。Western blot结果见图3,在75kDa大小处存在单一目的条带,与预期大小75kDa一致,表明CRP、SAA、PCT和IL6融合蛋白成功表达。
实施例4融合蛋白的纯化
(1)转染细胞72h后,将细胞超声破碎后,于4℃,12000rpm/min离心10min,留取上清液;
(2)将上清液于预先平衡好的Ni-NTA于4℃旋转孵育4h;
(3)用Washing Buffer(50mmol/L NaH2PO4,300mmol/L NaCl,50mmol/L咪唑)洗涤6遍,收集沉淀,之后与Elution buffer(50mmol/L NaH2PO4,300mmol/L NaCl,250mmol/L咪唑)于4℃缓慢混匀1h以洗脱目的蛋白。
(4)将上清放入透析袋中,置于冰冷的PBS溶液中进行透析,每隔1h换一次PBS,共换8次。最后透析袋中的蛋白溶液为纯化的蛋白质。通过SDS-PAGE鉴定纯化后的蛋白质,结果见图4。结果表明通过纯化后获得较纯的融合蛋白。
实施例5制备复合质控品进行性能测试
(1)效价验证
以实施例3和实施例4的方法获得的融合蛋白,以人血清作为基质(含有2%甘油及1%Proclin-300)按1/100、1/1000、1/10000配制SAA项目质控,使用SAA化学发光试剂盒进行检测,根据软件反算出复合质控原液浓度,结果如下表2所示:
表2复合质控浓度鉴定
试剂盒的线性范围在1-200μg/mL,根据稀释比例回算,反算原液效价在12500μg/mL左右。
(2)准确度验证
依照合适的浓度梯度稀释原液,分别配制炎症PCT和IL6项目复合质控及CRP和SAA项目复合质控;对上述配制复合质控品进行准确度分析,分别测量三次,计算均值与靶值的偏差。表3为质控品准确度分析数据。
表3质控品准确度数据分析
数据显示制备的复合质控品精密度及准确度都较好。
(3)稳定性验证
对复合质控效期稳定性进行考察。表4为随着时间延长,测得的结果与最初测定结果的计算偏差。
表4质控品稳定性数据分析
时间 | CRP | SAA | PCT | IL6 |
0月 | 0.82% | 0.52% | 1.09% | 0.63% |
4月 | 0.95% | 0.68% | 1.23% | 0.95% |
8月 | 0.91% | 0.75% | 2.21% | 1.10% |
12月 | 1.23% | 0.99% | 2.54% | 1.52% |
16月 | 1.28% | 1.52% | 3.21% | 1.58% |
20月 | 2.32% | 1.75% | 3.55% | 2.08% |
24月 | 2.15% | 1.95% | 4.02% | 3.11% |
数据显示制备的复合质控品在2年内稳定性较好。
本发明的炎症PCT和IL6项目复合质控及CRP和SAA项目复合质控,不仅具有较好的稳定性和准确性,同时能够有效降低质控品的成本,提高体外诊断检测的便捷性并能很好地控制质控品的批间差。
序列表
<110> 巴迪泰(广西)生物科技有限公司
<120> 融合表达蛋白及其制备方法和用途
<141> 2022-01-17
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 612
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atgtcgagga aggcttttgt gtttcccaaa gagtcggata cttcctatgt atccctcaaa 60
gcaccgttaa cgaagcctct caaagccttc actgtgtgcc tccacttcta cacggaactg 120
tcctcgaccc gtgggtacag tattttctcg tatgccacca agagacaaga caatgagatt 180
ctcatatttt ggtctaagga tataggatac agttttacag tgggtgggtc tgaaatatta 240
ttcgaggttc ctgaagtcac agtagctcca gtacacattt gtacaagctg ggagtccgcc 300
tcagggatcg tggagttctg ggtagatggg aagcccaggg tgaggaagag tctgaagaag 360
ggatacactg tgggggcaga agcaagcatc atcttggggc aggagcagga ttccttcggt 420
gggaactttg aaggaagcca gtccctggtg ggagacattg gaaatgtgaa catgtgggac 480
tttgtgctgt caccagatga gattaacacc atctatcttg gcgggccctt cagtcctaat 540
gtcctgaact ggcgggcact gaagtatgaa gtgcaaggcg aagtgttcac caaaccccag 600
ctgtggccct ga 612
<210> 2
<211> 369
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atgaagcttc tcacgggcct ggttttctgc tccttggtcc tgggtgtcag cagccgaagc 60
ttcttttcgt tccttggcga ggcttttgat ggggctcggg acatgtggag agcctactct 120
gacatgagag aagccaatta catcggctca gacaaatact tccatgctcg ggggaactat 180
gatgctgcca aaaggggacc tgggggtgcc tgggctgcag aagtgatcac cgatgccaga 240
gagaatatcc agagattctt tggccatggt gcggaggact cgctggctga tcaggctgcc 300
aatgaatggg gcaggagtgg caaagacccc aatcacttcc gacctgctgg cctgcctgag 360
aaatactga 369
<210> 3
<211> 426
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atgggcttcc aaaagttctc ccccttcctg gctctcagca tcttggtcct gttgcaggca 60
ggcagcctcc atgcagcacc attcaggtct gccctggaga gcagcccagc agacccggcc 120
acgctcagtg aggacgaagc gcgcctcctg ctggctgcac tggtgcagga ctatgtgcag 180
atgaaggcca gtgagctgga gcaggagcaa gagagagagg gctccagcct ggacagcccc 240
agatctaagc ggtgcggtaa tctgagtact tgcatgctgg gcacatacac gcaggacttc 300
aacaagtttc acacgttccc ccaaactgca attggggttg gagcacctgg aaagaaaagg 360
gatatgtcca gcgacttgga gagagaccat cgccctcatg ttagcatgcc ccagaatgcc 420
aactaa 426
<210> 4
<211> 639
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
atgaactcct tctccacaag cgccttcggt ccagttgcct tctccctggg gctgctcctg 60
gtgttgcctg ctgccttccc tgccccagta cccccaggag aagattccaa agatgtagcc 120
gccccacaca gacagccact cacctcttca gaacgaattg acaaacaaat tcggtacatc 180
ctcgacggca tctcagccct gagaaaggag acatgtaaca agagtaacat gtgtgaaagc 240
agcaaagagg cactggcaga aaacaacctg aaccttccaa agatggctga aaaagatgga 300
tgcttccaat ctggattcaa tgaggagact tgcctggtga aaatcatcac tggtcttttg 360
gagtttgagg tatacctaga gtacctccag aacagatttg agagtagtga ggaacaagcc 420
agagctgtgc agatgagtac aaaagtcctg atccagttcc tgcagaaaaa ggcaaagaat 480
ctagatgcaa taaccacccc tgacccaacc acaaatgcca gcctgctgac gaagctgcag 540
gcacagaacc agtggctgca ggacatgaca actcatctca ttctgcgcag ctttaaggag 600
ttcctgcagt ccagcctgag ggctcttcgg caaatgtag 639
<210> 5
<211> 6
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<213> 人工序列(Artificial Sequence)
<400> 5
aagctt 6
<210> 6
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<213> 人工序列(Artificial Sequence)
<400> 6
gccaccatgg 10
<210> 7
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
catcatcatc atcatcac 18
<210> 8
<211> 6
<212> DNA
<213> 人工序列(Artificial Sequence)
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tctaga 6
Claims (9)
1.一种表达单元,其特征在于,该表达单元从5’到3’依次为:Kozak基因-CRP基因-linker-SAA基因-linker-PCT基因-linker-IL6基因-组氨酸标签;所述的Kozak基因的核苷酸序列如SEQ ID NO:6所示,所述的CRP基因的核苷酸序列如SEQ ID NO:1所示,所述的SAA基因的核苷酸序列如SEQ ID NO:2所示,所述的PCT基因的核苷酸序列如SEQ ID NO:3所示,所述的IL6基因的核苷酸序列如SEQ ID NO:4所示;所述的linker的序列如下所示:EAAAKEAAAKEAAAK。
2.包含权利要求1所述的表达单元的重组载体。
3.根据权利要求2所述的重组载体,其特征在于:所述表达载体为pcDNA3.1。
4.包含权利要求1所述的表达单元、权利要求2或3所述的重组载体的宿主细胞。
5.根据权利要求4所述的宿主细胞,其特征在于:所述的宿主细胞为人HEK 293T细胞。
6.由权利要求1所述的表达单元、权利要求2或3所述的重组载体或权利要求4或5所述的宿主细胞表达得到的融合蛋白。
7.权利要求6所述的融合蛋白的制备方法,其特征在于,包括以下步骤:
a、筛选包含RP、SAA、PCT和IL6的目的基因,构建重组载体;
b、将步骤a得到的重组载体导入人HEK 293T细胞,经表达纯化后,得到融合蛋白。
8.根据权利要求7所述的融合蛋白的的制备方法,其特征在于,包括以下步骤:
a、依次合成获得CRP、SAA、PCT和IL6基因完整的CDS区,在基因之间插入刚性linker;在CRP基因起始密码子ATG前插入Hind III限制性内切酶酶切位点和Kozak序列,在IL6基因下游插入6*His标签及Xba I酶切位点;
b、将步骤a得到的目的基因与哺乳动物表达载体pcDNA3.1质粒通过Hind III和Xba I双酶切,用胶回收试剂盒纯化后用T4连接酶连接,经鉴定后获得重组载体;
c、将获得的重组载体瞬时转染至人HEK 293T,使其在细胞中转录翻译并得到同时表达CRP、SAA、PCT和IL6基因的融合蛋白质,72h后收集裂解细胞;
d、利用插入的His标签通过Ni-NTA纯化得到融合蛋白。
9.权利要求6所述的融合蛋白的用途,作为复合质控品用于制备炎症项目检测试剂盒。
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