CN114230643A - 一种SASR-Cov-2病毒S重组蛋白、细胞株及构建方法与应用 - Google Patents
一种SASR-Cov-2病毒S重组蛋白、细胞株及构建方法与应用 Download PDFInfo
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Abstract
本发明涉及生物技术领域,具体公开了一种SASR‑Cov‑2病毒S重组蛋白、细胞株及构建方法与应用。本发明利用SASR‑Cov‑2病毒S重组蛋白(SARS‑COV‑2 S(His))可以构建出高效特异性表达SASR‑Cov‑2病毒S重组蛋白的急性肺损伤细胞株,其构建方法稳定性好,重现性高;其构建得到的细胞株为针对性筛选新型冠状病毒肺炎(COVID‑19)治疗药物提供细胞株模型。
Description
技术领域
本发明涉及生物技术领域,尤其是涉及一种SASR-Cov-2病毒S重组蛋白、细胞株及构建方法与应用。
背景技术
急性肺损伤(Acute lung injury,ALI)是临床常见急重症,以进行性加重的呼吸困难、顽固的低氧血症为临床特征,其严重阶段可发展为成人呼吸窘迫综合征(Acuterespiratory distress syndrome,ARDS),ARDS是临床危重症发生急性呼吸衰竭的主要原因,病死率高达50%。常见的致病因素主要有:感染、有害物质吸入、外伤、休克、中毒等。
有研究证明SARS-Cov-2诱导的急性肺损伤(ALI)是新型冠状病毒肺炎(COVID-19)患者死亡的主要病因,其机制为SARS-Cov-2与细胞受体血管紧张素转换酶2(ACE2)结合,导致ACE2缺失及其反馈性诱导血管紧张素Ⅱ(AngⅡ)和血管紧张素Ⅰ型受体(AT1R)介导的急性炎症反应。肺泡II型上皮细胞作为肺泡主要结构细胞,在炎症因子和氧化应激的攻击下,其凋亡或坏死的数量直接决定了ALI的严重程度及预后走向,ALI/ARDS缺乏特效治疗,因而其发病机制以及药物筛选是本领域内的研究热点。
由于急性肺损伤(ALI)的发病机制较为复杂,其病理基础是肺内失控的炎症反应所致的肺泡毛细血管膜损伤,进而导致肺水肿及透明膜形成。常规方法构建的急性肺损伤细胞模型,在面对新型冠状病毒肺炎(COVID-19)筛选治疗药物其特异性、灵敏度较低,较难准确的筛选出治疗新型冠状病毒肺炎(COVID-19)效果好、灵敏度高的药物成分或药物。
发明内容
本发明的目的在于克服上述现有技术的不足之处而提供一种SASR-Cov-2病毒S重组蛋白、急性肺损伤细胞株及构建方法与应用,本发明利用SASR-Cov-2病毒S重组蛋白可以构建出具有特异性的急性肺损伤的细胞株,为针对性筛选新型冠状病毒肺炎(COVID-19)治疗药物提供细胞株模型,特异性强,灵敏度高。
为实现上述目的,本发明采取的技术方案为:
第一目的,本发明提供了一种SASR-Cov-2病毒S重组蛋白,所述SASR-Cov-2病毒S重组蛋白的序列如SEQ ID NO:1所示,或与SEQ ID NO:1相比具有一个或几个氨基酸的置换、缺失或添加的序列组成。
第二目的,本发明提供了一种检测SASR-Cov-2病毒S重组蛋白的引物,所述引物包括如SEQ ID NO:2~5所示的序列。
第三目的,本发明提供了一种基因,所述基因编码上述SASR-Cov-2病毒S重组蛋白。
第四目的,本发明提供了一种蛋白质表达载体,所述蛋白质表达载体携带上述基因。
第五目的,本发明提供了一种细胞株,所述细胞株携带上述的基因或蛋白质表达载体。
第六目的,本发明提供了一种急性损伤细胞株的构建方法,将上述的蛋白质表达载体进行转染及扩大培养,构建得到含有SASR-Cov-2病毒S重组蛋白的细胞株;通过逆转录、PCR和Western Blot对细胞株表达SASR-Cov-2病毒S重组蛋白的基因进行验证。
作为本发明所述急性损伤细胞株的构建方法的优选实施方式,所述细胞株包括Vero E6细胞株。
作为本发明所述急性损伤细胞株的构建方法的优选实施方式,转染的步骤为:用无血清培养基稀释蛋白质表达载体,用相同的无血清培养基稀释Hieff Trans TM试剂;静置,涡旋表达载体溶液,并添加稀释后的Hieff Trans TM试剂,混匀静置,形成DNA-HieffTrans TM复合物;然后将DNA-Hieff Trans TM复合物加入孔中,继续培养,收集细胞株。
第六七目的,本发明提供了上述细胞株在筛选新型冠状病毒肺炎治疗药物中的应用。
本发明通过生物学手段,将携带SASR-Cov-2病毒S重组蛋白(SARS-COV-2S(His))的蛋白质表达载体进行转染及扩大培养实验,构建表达SASR-Cov-2病毒S重组蛋白的VeroE6细胞株。并通过逆转录、PCR和western blot等实验方法验证细胞株表达SASR-Cov-2病毒S重组蛋白的有效性。
与现有技术相比,本发明具有以下有益效果:
本发明利用SASR-Cov-2病毒S重组蛋白(SARS-COV-2S(His))可以构建出高效特异性表达SASR-Cov-2病毒S重组蛋白的急性肺损伤细胞株,其构建方法稳定性好,重现性高;其构建得到的细胞株为针对性筛选新型冠状病毒肺炎(COVID-19)治疗药物提供细胞株模型。
附图说明
图1为SASR-Cov-2病毒S重组蛋白的PCR结果图;
图2为本发明构建的蛋白质表达载体的图谱;
图3为SASR-Cov-2病毒S重组蛋白的鉴定结果图;
图4为GAPDH的PCR曲线图I;
图5为GAPDH的PCR曲线图II;
图6为GAPDH的PCR曲线图III;
图7为SASR-Cov-2病毒S重组蛋白的PCR曲线图I;
图8为SASR-Cov-2病毒S重组蛋白的PCR曲线图II;
图9为SASR-Cov-2病毒S重组蛋白的PCR曲线图III;
图10为空载组和SASR-Cov-2病毒S重组蛋白过表达组表达mRNA的结果图;
图11为anti-GAPDH(36kPa)的western blot结果图;
图12为anti-His-SASR-Cov-2病毒S重组蛋白(142kPa)的western blot结果图。
具体实施方式
为更好的说明本发明的目的、技术方案和优点,下面将结合附图和具体实施例对本发明作进一步说明。
在以下实施例中,所使用的实验方法如无特殊说明,均为常规方法,所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
SASR-Cov-2病毒S重组蛋白、引物信息如表1和表2所示。
表1
基因名称 | 基因ID | 转录本ID | 产物 | 引物位置 |
SASR-Cov-2病毒S重组蛋白 | 43740568 | NC-045512.2 | 3861bp | 1-3861 |
SASR-Cov-2病毒S重组蛋白 | 43740568 | NC-045512.2 | 1369bp | 794-2162 |
GAPDH | 14433 | NM-001289726.1 | 150bp | 948-197 |
表2
实施例1、构建蛋白质表达载体
通过基因重组技术构建携带编码SASR-Cov-2病毒S重组蛋白的基因的蛋白质表达载体,具体包括以下步骤:
1)PCR:①PCR扩增体系:取重组蛋白模板2μL;引物各2μL;Hieff Canace GoldHigh-Fidelity DNA Polymerase(2U/μL)1μL;2×Canace Gold PCR buffer(含Mg2+,dNTPs)25μL;ddH2O 18μL;
②PCR扩增程序:预变性98℃/3min;30个循环(变性98℃/10s,退火65℃/20s,延伸72℃1kb/min),延伸72℃/5min,12℃保存。PCR结果见图1所述。
2)质粒提取:①空载菌落接种于5mL含有相应抗性的LB培养基(GL7002,捷瑞)中并置于恒温摇床中37℃、220rpm震荡摇晃过夜培养;②使用质粒DNA微量提取试剂盒提取质粒。
3)酶切:①酶切体系:pLVX-AcGFP1-N1 900-1500ng;Fastdigest EcoR I(货号FD0274,品牌Thermo fisher)1μL;Fastdigest Xho I(FD0694,品牌Thermo fisher)1μL;10×universe Buffer 5μL;ddH2O To 50μL;
②酶切程序:37℃,10~30min。
4)同源重组:①配制同源重组体系:120ng酶切质粒、100ng PCR片段、5μL 2×Assembly Mix、补足ddH2O TO 10μL;
②反应体系:轻轻混合,50℃连接15分钟,反应结束后,将离心管置于冰上冷却数秒之后可将重组产物(蛋白质表达载体)保存于-20℃或直接用于转化。其载体的图谱如图2所示。
4)感受态细胞转化:
①连接体系与50μL感受态细胞混合,冰上孵育30min;②42℃热激45s;③瞬间转移至冰上孵育5min;④将感受态细胞与1mL LB培养基混合后37℃震荡培养45min;⑤涂板。
5)鉴定:①挑取培养板上的单菌落(5-10个),溶于10μL的无菌水中,取4μL作为模板进行PCR;②剩下的加入1mL含相应抗性的培养基中37℃摇床培养。鉴定结果见图3,鉴定引物为F:P10077和R:P10078,产物长度为1369bp。
6)测序:①PCR鉴定阳性菌落,送交测序公司,使用测序引物开展一代测序。测定SASR-Cov-2病毒S重组蛋白序列如SEQ ID NO:1所示,重组蛋白大小为142.9kDa。
7)无内毒素质粒提取:
①取5mL过夜培养的菌液,12000×g离心2min收集菌体取过夜培养的菌液,去上清(尽量吸尽)。
②如菌液量过大,可分多次离心收集;
③加入无色溶液RB(含RNase A)250μL,振荡悬浮菌体,不应留有小的菌块;
④加入蓝色溶液LB 250μL,温和地上下翻转混合4-6次,使菌体充分裂解,形成蓝色透亮的溶液,颜色由半透亮变为透亮蓝色,指示完全裂解(不宜超过5分钟);
⑤加入黄色溶液NB 350μL,轻轻混合5-6次(颜色由蓝色完全变成黄色,指示混合均匀,中和完全)直至形成紧实的黄色凝集块,室温静置2分钟;
⑥12000xg离心5分钟,小心吸取上清加入高心柱中。12000xg离心1分钟,弃流出液(如上清体积大于800μL,可以分成多次加入柱中,并同上离心,弃流出液。);
⑦加入250μL溶液TB,室温静置10分钟,12000xg离心1分钟,弃流出液;
⑧加入650μL溶液WB,12000xg离心1分钟,弃流出液;
⑨12000xg离心1-2分钟,彻底去除残留的WB;
⑩将离心柱置于一干净的离心管中,在柱的中央加入50-70μL EB或去离子水(pH>7.0)室温静置1分钟。(EB或去离子水在60-70℃水浴预热,使用效果更好)。10000g离心1分钟,洗脱DNA,洗脱出的DNA于-20℃保存。
实验结果:
经过PCR及鉴定步骤,测得PCR条带大小与目的扩增片段相同,代表该克隆为载体重组成功克隆。
实施例2、急性肺损伤细胞株的构建方法
1、核酸转染、扩大培养
种板:转染前一天,将Vero E6细胞株接种细胞培养板,控制第二天细胞汇合度约为70-80%;
温度平衡:将Hieff TransTM、无内毒素质粒、无血清培养基平衡至15-25℃;
配置转染体系:用50μl无血清培养基稀释2μg蛋白质表达载体,用同样的50μl培养基稀释6μl Hieff TransTM试剂;静置5min,轻轻涡旋载体溶液,并逐滴添加稀释的HieffTransTM试剂,充分混匀后,室温精制10-25min以形成DNA-Hieff TransTM复合物;
转染:把培养板中的旧培养基换成1ml新鲜基础培养基,并将复合物逐滴加入孔中,边加边摇晃,37℃,5%CO2培养基继续培养48h,收集细胞。
2、逆转录
样本前处理:Vero E6细胞株用预冷1ml 1×PBS重悬,1600g离心3-5min,弃去上清液;每107细胞的量加1ml Magzol。
总RNA提取:①将1.5ml离心管剧烈震荡10次,室温孵育5min;②按0.2ml氯仿/1mlMagzol加入氯仿并充分震荡20s;③12000g,4℃离心10min;④吸取上清(0.5ml上清/1mlMagzol)转移至新离心管中,切勿吸到中间层;⑤按等倍体积加入异丙醇,充分混匀;⑥冰上静置15-20min;⑦12000g,4℃离心10min,小心弃去上清;⑧加入1ml预冷的75%乙醇(无酶水配制),7500g,4℃离心5min,小心弃去上清;⑨打开EP管盖,室温自然挥发乙醇10min;⑩加入30-50μl DEPC溶液室温溶解RNA 5min。
质量检测:①取2μl RNA样品检测RNA OD260、OD280,根据OD260/OD280计算总RNA浓度及杂质污染程度;②剩余RNA样品置于-80℃保存。
cDNA合成:①逆转录体系:4×gDNA wiper Mix:4μl、总RNA:0.5-1μl,加RNasefree ddH2O补齐至16μl,42℃2min;②加5×HiScript II qRT SuperMix II:4μl,37℃15min、85℃5s、12℃终止;③-20℃保存cDNA待用。
3、PCR
qPCR:①反应体系配制:ChamQ Universal SYBR qPCR(2×):10μl、ForwardPrimer(F:P10015,10μM):1μL、Reverse Primer(R:P10016,10μM):1μL、cDNA模板:2μL、ddH2O补齐至20μL;②95℃预变性5min;③40个循环:95℃变性15s、60℃退火20s、72℃延伸20s;采集荧光信号;④70-95℃变性溶解曲线。
数据分析:
①△Ct公式为:△Ct=Ct(目的基因)-Ct(内参基因);②△△Ct公式为:△△Ct=△Ct(目标样本)-△Ct(对照样本);③2e(-△△Ct):以2为底,以-△△Ct为幂值,结果呈现差异倍数。
结果参考图4-10(图4-6为GAPDH(内参基因)的PCR曲线图,图7-9为SASR-Cov-2病毒S重组蛋白的PCR曲线图,图10为空载组和SASR-Cov-2病毒S重组蛋白过表达组表达mRNA的结果图)。
4、western blot
蛋白质样品制备:①用1×PBS清洗细胞3次,加入适量裂解液(6孔板每孔加60-100μL),样品冰上裂解30min;②4℃,12000rpm离心5min,转移上清到新的EP管中;③向样品中加入5×Loading buffer稀释至1×,沸水浴10min后置于冰上或-20℃保存。
蛋白浓度测定:①方法一:取2ml考马斯亮蓝G250与1ml水混合,加入2μL蛋白样品,可见光分光光度计595nm测吸光度;方法二:使用BCA试剂盒测定蛋白浓度;方法三:使用浓度检测仪进行检测;②根据吸光度值计算蛋白浓度,用裂解液调整蛋白浓度使其在一个水平。
蛋白质量检测:①蛋白样品加入含5%β-巯基乙醇的5×蛋白上样缓冲液(样品:缓冲液=4:1),沸水煮10min变性;②取10μL蛋白样品跑凝胶电泳,制备好的样品放-20℃保存;③考马斯亮蓝G250染色,摇床上摇30min;④加水放微波炉中高火脱色5min,2-3次。
聚丙烯酰胺凝胶电泳:①制备分离胶和浓缩胶,分离胶的浓度根据蛋白分子量而定(分子量10-50kDa配12%,分子量50-80kDa配8%)。②蛋白上样量每孔约50μg或15-20μL,蛋白Marker上3μL。③首先电压为80V电泳15min,随后更改电压为120V继续电泳40min。
转膜:①根据胶的大小裁剪PVDF膜(8个样全膜可裁5cm×8cm),剪角以分反正,然后将膜浸泡在甲醇中活化;②转模夹板黑色朝下,依次放海绵、滤纸(3层)、胶、PVDF膜、滤纸(3层)、海绵;③胶根据目的蛋白分子量裁切;④夹好夹板,在转模槽内放一个冰盒,加满转模缓冲液,然后置于冰上转模30min。
封闭:①转模结束后,取出膜,正面朝上,用1×TBST洗涤2-3次,每次5min。②加入封闭液(5%脱脂牛奶或4%BSA),4℃过夜或室温摇床摇2-3h封闭。
抗体孵育:①封闭结束后,膜用1×TBST洗涤6次,每次5min;②参考抗体说明书确定稀释比稀释一抗,用封膜机和PE手套将膜封起来,室温摇床摇2h;③回收一抗,膜用1×TBST洗涤3次,每次10min;④按抗体说明书稀释二抗,用封膜机和PE手套将膜封起来,室温摇床摇1h;⑤回收二抗,膜用1×TBST洗涤3次,每次10min。
显影:①超敏发光液A、B等量混合,1张膜需200μL;在膜上末端条带处均匀滴加发光液,置于曝光仪器内自动显影。
结果解读:蛋白含量在不同样品间的实际变化结果,图11为anti-GAPDH(36kPa)的western blot结果图(1为空载组、2为SASR-Cov-2病毒S重组蛋白过表达组、3为SASR-Cov-2病毒S重组蛋白过表达组(上清));
图12为anti-His-SASR-Cov-2病毒S重组蛋白(142kPa)的western blot结果图(1为空载组、2为SASR-Cov-2病毒S重组蛋白过表达组、3为SASR-Cov-2病毒S重组蛋白过表达组(上清))。
最后所应当说明的是,以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制,尽管参照较佳实施例对本发明作了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的实质和范围。
SEQUENCE LISTING
<110> 东莞市松山湖中心医院(东莞市石龙人民医院、东莞市第三人民医院、东莞市心血管病研究所)
<120> 一种SASR-Cov-2病毒S重组蛋白、细胞株及构建方法与应用
<130> 2021.12.21
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 3861
<212> DNA
<213> 人工合成
<400> 1
atgggccatc atcaccatca ccattttgtt tttcttgttt tattgccact agtctctagt 60
cagtgtgtta atcttacaac cagaactcaa ttaccccctg catacactaa ttctttcaca 120
cgtggtgttt attaccctga caaagttttc agatcctcag ttttacattc aactcaggac 180
ttgttcttac ctttcttttc caatgttact tggttccatg ctatacatgt ctctgggacc 240
aatggtacta agaggtttga taaccctgtc ctaccattta atgatggtgt ttattttgct 300
tccactgaga agtctaacat aataagaggc tggatttttg gtactacttt agattcgaag 360
acccagtccc tacttattgt taataacgct actaatgttg ttattaaagt ctgtgaattt 420
caattttgta atgatccatt tttgggtgtt tattaccaca aaaacaacaa aagttggatg 480
gaaagtgagt tcagagttta ttctagtgcg aataattgca cttttgaata tgtctctcag 540
ccttttctta tggaccttga aggaaaacag ggtaatttca aaaatcttag ggaatttgtg 600
tttaagaata ttgatggtta ttttaaaata tattctaagc acacgcctat taatttagtg 660
cgtgatctcc ctcagggttt ttcggcttta gaaccattgg tagatttgcc aataggtatt 720
aacatcacta ggtttcaaac tttacttgct ttacatagaa gttatttgac tcctggtgat 780
tcttcttcag gttggacagc tggtgctgca gcttattatg tgggttatct tcaacctagg 840
acttttctat taaaatataa tgaaaatgga accattacag atgctgtaga ctgtgcactt 900
gaccctctct cagaaacaaa gtgtacgttg aaatccttca ctgtagaaaa aggaatctat 960
caaacttcta actttagagt ccaaccaaca gaatctattg ttagatttcc taatattaca 1020
aacttgtgcc cttttggtga agtttttaac gccaccagat ttgcatctgt ttatgcttgg 1080
aacaggaaga gaatcagcaa ctgtgttgct gattattctg tcctatataa ttccgcatca 1140
ttttccactt ttaagtgtta tggagtgtct cctactaaat taaatgatct ctgctttact 1200
aatgtctatg cagattcatt tgtaattaga ggtgatgaag tcagacaaat cgctccaggg 1260
caaactggaa agattgctga ttataattat aaattaccag atgattttac aggctgcgtt 1320
atagcttgga attctaacaa tcttgattct aaggttggtg gtaattataa ttacctgtat 1380
agattgttta ggaagtctaa tctcaaacct tttgagagag atatttcaac tgaaatctat 1440
caggccggta gcacaccttg taatggtgtt gaaggtttta attgttactt tcctttacaa 1500
tcatatggtt tccaacccac taatggtgtt ggttaccaac catacagagt agtagtactt 1560
tcttttgaac ttctacatgc accagcaact gtttgtggac ctaaaaagtc tactaatttg 1620
gttaaaaaca aatgtgtcaa tttcaacttc aatggtttaa caggcacagg tgttcttact 1680
gagtctaaca aaaagtttct gcctttccaa caatttggca gagacattgc tgacactact 1740
gatgctgtcc gtgatccaca gacacttgag attcttgaca ttacaccatg ttcttttggt 1800
ggtgtcagtg ttataacacc aggaacaaat acttctaacc aggttgctgt tctttatcag 1860
gatgttaact gcacagaagt ccctgttgct attcatgcag atcaacttac tcctacttgg 1920
cgtgtttatt ctacaggttc taatgttttt caaacacgtg caggctgttt aataggggct 1980
gaacatgtca acaactcata tgagtgtgac atacccattg gtgcaggtat atgcgctagt 2040
tatcagactc agactaattc tcctcggcgg gcacgtagtg tagctagtca atccatcatt 2100
gcctacacta tgtcacttgg tgcagaaaat tcagttgctt actctaataa ctctattgcc 2160
atacccacaa attttactat tagtgttacc acagaaattc taccagtgtc tatgaccaag 2220
acatcagtag attgtacaat gtacatttgt ggtgattcaa ctgaatgcag caatcttttg 2280
ttgcaatatg gcagtttttg tacacaatta aaccgtgctt taactggaat agctgttgaa 2340
caagacaaaa acacccaaga agtttttgca caagtcaaac aaatttacaa aacaccacca 2400
attaaagatt ttggtggttt taatttttca caaatattac cagatccatc aaaaccaagc 2460
aagaggtcat ttattgaaga tctacttttc aacaaagtga cacttgcaga tgctggcttc 2520
atcaaacaat atggtgattg ccttggtgat attgctgcta gagacctcat ttgtgcacaa 2580
aagtttaacg gccttactgt tttgccacct ttgctcacag atgaaatgat tgctcaatac 2640
acttctgcac tgttagcggg tacaatcact tctggttgga cctttggtgc aggtgctgca 2700
ttacaaatac catttgctat gcaaatggct tataggttta atggtattgg agttacacag 2760
aatgttctct atgagaacca aaaattgatt gccaaccaat ttaatagtgc tattggcaaa 2820
attcaagact cactttcttc cacagcaagt gcacttggaa aacttcaaga tgtggtcaac 2880
caaaatgcac aagctttaaa cacgcttgtt aaacaactta gctccaattt tggtgcaatt 2940
tcaagtgttt taaatgatat cctttcacgt cttgacaaag ttgaggctga agtgcaaatt 3000
gataggttga tcacaggcag acttcaaagt ttgcagacat atgtgactca acaattaatt 3060
agagctgcag aaatcagagc ttctgctaat cttgctgcta ctaaaatgtc agagtgtgta 3120
cttggacaat caaaaagagt tgatttttgt ggaaagggct atcatcttat gtccttccct 3180
cagtcagcac ctcatggtgt agtcttcttg catgtgactt atgtccctgc acaagaaaag 3240
aacttcacaa ctgctcctgc catttgtcat gatggaaaag cacactttcc tcgtgaaggt 3300
gtctttgttt caaatggcac acactggttt gtaacacaaa ggaattttta tgaaccacaa 3360
atcattacta cagacaacac atttgtgtct ggtaactgtg atgttgtaat aggaattgtc 3420
aacaacacag tttatgatcc tttgcaacct gaattagact cattcaagga ggagttagat 3480
aaatatttta agaatcatac atcaccagat gttgatttag gtgacatctc tggcattaat 3540
gcttcagttg taaacattca aaaagaaatt gaccgcctca atgaggttgc caagaattta 3600
aatgaatctc tcatcgatct ccaagaactt ggaaagtatg agcagtatat aaaatggcca 3660
tggtacattt ggctaggttt tatagctggc ttgattgcca tagtaatggt gacaattatg 3720
ctttgctgta tgaccagttg ctgtagttgt ctcaagggct gttgttcttg tggatcctgc 3780
tgcaaatttg atgaagacga ctctgagcca gtgctcaaag gagtcaaatt acattacaca 3840
catcatcacc atcaccattg a 3861
<210> 2
<211> 88
<212> DNA
<213> 人工合成
<400> 2
gctaccggac tcagactcga ggccaccatg ggccatcatc accatcacca ttttgttttt 60
cttgttttat tgccactagt ctctagtc 88
<210> 3
<211> 77
<212> DNA
<213> 人工合成
<400> 3
ggtaccgtcg actgcatgaa ttctcaatgg tgatggtgat gatgtgtgta atgtaatttg 60
actcctttga gcactgg 77
<210> 4
<211> 21
<212> DNA
<213> 人工合成
<400> 4
ggacagctgg tgctgcagct t 21
<210> 5
<211> 29
<212> DNA
<213> 人工合成
<400> 5
atggcaatag agttattaga gtaagcaac 29
Claims (9)
1.一种SASR-Cov-2病毒S重组蛋白,其特征在于,所述SASR-Cov-2病毒S重组蛋白的序列如SEQ ID NO:1所示,或与SEQ ID NO:1相比具有一个或几个氨基酸的置换、缺失或添加的序列组成。
2.一种检测SASR-Cov-2病毒S重组蛋白的引物,其特征在于,所述引物包括如SEQ IDNO:2~5所示的序列。
3.一种基因,其特征在于,所述基因编码如权利要求1所述的SASR-Cov-2病毒S重组蛋白。
4.一种蛋白质表达载体,其特征在于,所述蛋白质表达载体携带如权利要求3所述的基因。
5.一种细胞株,其特征在于,所述细胞株携带如权利要求3所述的基因或权利要求4所述的蛋白质表达载体。
6.一种急性肺损伤细胞株的构建方法,其特征在于,将如权利要求4所述的蛋白质表达载体进行转染及扩大培养,构建得到含有SASR-Cov-2病毒S重组蛋白的细胞株;通过逆转录、PCR和Western Blot对细胞株表达SASR-Cov-2病毒S重组蛋白的基因进行验证。
7.如权利要求6所述的构建方法,其特征在于,所述细胞株包括Vero E6细胞株。
8.如权利要求6所述的构建方法,其特征在于,转染的步骤为:用无血清培养基稀释蛋白质表达载体,用相同的无血清培养基稀释Hieff Trans TM试剂;静置,涡旋表达载体溶液,并添加稀释后的Hieff Trans TM试剂,混匀静置,形成DNA-Hieff Trans TM复合物;然后将DNA-Hieff Trans TM复合物加入孔中,继续培养,收集细胞株。
9.如权利要求5所述的细胞株在筛选新型冠状病毒肺炎治疗药物中的应用。
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