CN115073564A - 一种新型冠状病毒的基因工程重组蛋白、重组载体、重组工程菌和疫苗 - Google Patents
一种新型冠状病毒的基因工程重组蛋白、重组载体、重组工程菌和疫苗 Download PDFInfo
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Abstract
本发明提供了一种新型冠状病毒的基因工程重组蛋白、重组载体、重组工程菌和疫苗，涉及疫苗技术领域。本发明所述重组蛋白以SARS‑CoV‑2棘突蛋白(spike，S)RBD区域Val308‑Gly548作为抗原靶点，并对其编码基因进行密码子优化得到，并基于所述重组蛋白建立了重组表达载体和原核表达系统，利用生产得到的重组蛋白构建重组亚单位蛋白疫苗。
Description
技术领域
本发明属于疫苗技术领域，具体涉及一种新型冠状病毒的基因工程重组 蛋白、重组载体、重组工程菌和疫苗。
背景技术
新型冠状病毒肺炎(Corona Virus Disease 2019,COVID-19)，简称“新冠 肺炎”，是指新型冠状病毒(Severe Acute Respiratory Syndrome Coronavirus 2, SARS-CoV-2)感染导致的肺炎。为了有效控制疫情，在全人群中推广接种 安全高效的新冠疫苗也逐渐成为了所有国家的共识。
目前在我国上市并使用的新型冠状病毒疫苗有很多种，按照其制备原理 大致可分为灭活疫苗，腺病毒载体疫苗，基因工程重组蛋白疫苗以及核酸疫 苗。其中，首个获批的基因工程重组蛋白疫苗是由安徽智飞龙科马生物制药 有限公司与中国科学院微生物研究所合作研发的，该疫苗的安全性高，量产 流程比较成熟，对整个生产线的生物安全防护要求也不高，十分适合短时间 大批量生产，弥补我国现阶段的新冠疫苗供需缺口。但是，基因工程重组蛋 白疫苗的缺陷在于其免疫原性较弱，需要加强免疫多次才能达到比较好的免疫效果，如何提高疫苗的免疫效果成为一个新的研究课题。
发明内容
有鉴于此，本发明的目的在于提供一种新型冠状病毒的基因工程重组蛋 白、重组载体、重组工程菌和疫苗，可解决基因工程重组蛋白疫苗存在的免 疫原性较弱的缺陷，安全有效、制备便捷、能够极大提高现有基因工程重组 蛋白疫苗免疫效果。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了一种新型冠状病毒SARS-CoV-2的基因工程重组蛋白，所 述重组蛋白的编码基因如SEQ ID NO.1所示。
优选的，所述重组蛋白的氨基酸序列如SEQ ID NO.2所示。
本发明还提供了一种表达上述重组蛋白的重组载体，所述重组载体以原 核表达载体为基础载体。
优选的，当以pET28a(+)为基础载体时，所述重组蛋白的编码基因插 入所述基础载体的NcoI和XhoI酶切位点之间。
本发明还提供了一种表达上述重组蛋白的重组工程菌，所述重组工程菌 以原核细胞为基础细胞。
本发明还提供了上述重组蛋白或利用上述重组工程菌生产得到的重组 蛋白在制备新型冠状病毒SARS-CoV-2重组亚单位蛋白疫苗中的应用。
本发明还提供了一种新型冠状病毒SARS-CoV-2重组亚单位蛋白疫苗， 包括上述重组蛋白或利用上述重组工程菌生产得到的重组蛋白和佐剂，所述 佐剂包括壳聚糖。
优选的，所述重组蛋白和佐剂的体积比为9:1。
本发明还提供了上述重组亚单位蛋白疫苗的制备方法，将壳聚糖溶于食 用醋酸中，冷却后作为佐剂；
将上述重组蛋白或利用上述重组工程菌生产得到的重组蛋白稀释至 0.5mg/mL，得亚单位蛋白；
将所述亚单位蛋白与佐剂按照9:1的体积比混合，得所述重组亚单位蛋 白疫。
有益效果：本发明提供了一种新型冠状病毒SARS-CoV-2的基因工程重 组蛋白，所述重组蛋白以SARS-CoV-2棘突蛋白(spike，S)RBD区域Val308 -Gly548作为抗原靶点，并对其编码基因进行密码子优化得到，并基于所述 重组蛋白建立了重组表达载体和原核表达系统，利用生产得到的重组蛋白构 建重组亚单位蛋白疫苗。
附图说明
图1为喷雾接种的模式图。
具体实施方式
本发明提供了一种新型冠状病毒SARS-CoV-2的基因工程重组蛋白，所 述重组蛋白的编码基因如SEQ ID NO.1所示。
本发明所述重组蛋白优选以S蛋白RBD区域Val308-Gly548(SEQ ID NO.2)为原始蛋白，同时以所述Val308-Gly548区域作为抗原靶点。在本发 明中，SARS-CoV-2棘突蛋白(spike，S)在空间结构上位于病毒包膜外表 面，负责与宿主细胞表面的ACE(Angiotensin-Converting Enzyme)受体识 别并结合，是病毒入侵进入宿主细胞的关键蛋白。
本发明按照氨基酸编码法则，将SEQ ID NO.2所示的氨基酸序列 (VEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVY AWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSN NLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGV EGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTG)转换成核酸序列，同时对所示氨基酸 序列的编码基因进行密码子优化，最终得到SEQ ID NO.1所示的序列： CCATGGTTGA GAAAGGTATC TACCAGACCT CTAACTTCCGTGTTCAGCCGACCGAATCTATCGTTCGTTTCCCGAACAT CACCAACCTGTGCCCGTTCGGTGAAGTGTTCAACGCTACCCGTTTCGC TTCTGTTTACGCTTGGAACCGTAAACGTATCTCTAACTGCGTTGCTGAC TACTCTGTTCTGTACAACTCTGCTAGCTTCTCTACCTTCAAATGCTACGG TGTTAGTCCGACCAAACTGAACGACCTGTGCTTCACCAACGTTTACGC TGACAGCTTCGTTATCCGTGGTGACGAAGTTCGTCAGATCGCTCCGGG TCAGACCGGTAAGATCGCTGACTACAACTACAAACTGCCGGACGACTT CACCGGTTGCGTTATCGCTTGGAACTCTAATAACCTGGACTCTAAAGTT GGTGGTAACTACAACTACCTGTACCGTCTGTTCCGTAAATCTAACCTGA AACCGTTCGAACGTGACATCTCTACCGAAATCTACCAGGCTGGTTCTAC TCCGTGCAACGGTGTTGAAGGTTTCAACTGCTACTTCCCGCTGCAGTC TTACGGTTTCCAGCCGACCAACGGTGTTGGTTACCAGCCGTACCGTGT TGTGGTTCTGAGCTTTGAACTGCTGCACGCTCCGGCTACTGTTTGCGGT CCGAAGAAATCTACCAACCTGGTTAAGAACAAATGCGTTAACTTCAAC TTCAACGGTCTGACTGGTGGCGGTCAGTACATCAAAGCTAACTCTAAA TT CATCGGTATCTTCGAATAACTCGAG，以促进在重组工程菌中的表达 量。
本发明还提供了一种表达上述重组蛋白的重组载体，所述重组载体以原 核表达载体为基础载体。
本发明所述原核表达载体优选包括pET28a(+)，当以pET28a(+)为 基础载体时，所述重组蛋白的编码基因优选插入所述基础载体的NcoI和 XhoI酶切位点之间。
本发明对所述重组载体的构建方法并没有特殊限定，利用双酶切或人工 合成的方法构建均可，本发明实施例中以双酶切的方式构建，但是不能仅将 其认定为本发明的全部保护范围。
本发明还提供了一种表达上述重组蛋白的重组工程菌，所述重组工程菌 以原核细胞为基础细胞。
本发明所述原核细胞优选包括大肠杆菌细胞。
本发明对所述重组工程菌的制备方法并没有特殊限定，优选利用转化的 方法，本发明所述转化优选包括电转化或氯化钙法转化。
在本发明实施例中，优选利用构建成功的表达载体经氯化钙法转化进入 大肠杆菌感受态细胞，抗生素筛选后将转化成功的大肠杆菌菌株接种到LB 液体培养基中，经IPTG诱导后分离并收集菌体沉淀，菌体沉淀中包含所述 重组蛋白，而后经分离和纯化得所述重组蛋白。
本发明还提供了上述重组蛋白或利用上述重组工程菌生产得到的重组 蛋白在制备新型冠状病毒SARS-CoV-2重组亚单位蛋白疫苗中的应用。
本发明还提供了一种新型冠状病毒SARS-CoV-2重组亚单位蛋白疫苗， 包括上述重组蛋白或利用上述重组工程菌生产得到的重组蛋白和佐剂，所述 佐剂包括壳聚糖。
本发明所述重组亚单位蛋白疫苗中，重组蛋白和佐剂的体积比优选为 9:1。
本发明还提供了上述重组亚单位蛋白疫苗的制备方法，将壳聚糖溶于食 用醋酸中，冷却后作为佐剂；
将上述重组蛋白或利用上述重组工程菌生产得到的重组蛋白稀释至 0.5mg/mL，得亚单位蛋白；
将所述亚单位蛋白与佐剂按照9:1的体积比混合，得所述重组亚单位蛋 白疫。
本发明优选将壳聚糖溶于体积百分含量为1％的食用醋酸中，并通过加 热的方法进行溶解，而后冷却，作为佐剂。实施例中，优选将2g壳聚糖放 入100mL 1％的食用醋酸中，100℃加热溶解；冷却后作为佐剂，4℃保存。
下面结合实施例对本发明提供的一种新型冠状病毒的基因工程重组蛋 白、重组载体、重组工程菌和疫苗进行详细的说明，但是不能把它们理解为 对本发明保护范围的限定。
实施例1
一种新型冠状病毒RBD基因工程重组蛋白亚单位疫苗的制备
(1)目的蛋白的选取
SARS-CoV-2棘突蛋白(spike，S)在空间结构上位于病毒包膜外表面， 负责与宿主细胞表面的ACE(Angiotensin-Converting Enzyme)受体识别并 结合，是病毒入侵进入宿主细胞的关键蛋白，本发明选取S蛋白RBD区域 Val308-Gly548(SEQ ID NO.2)作为抗原靶点。
(2)目的基因的合成与克隆
目的蛋白的编码基因进行密码子优化，优化后的基因序列(SEQ ID NO.1)交由公司合成。之后采用限制性内切酶NcoI和XhoI进行酶切处理， 酶切产物回收后连接到pET28a(+)表达载体上。
(3)目的蛋白的表达和前期处理
构建成功的表达载体经氯化钙法转化进入大肠杆菌感受态细胞，抗生素 筛选后将转化成功的大肠杆菌菌株接种到LB液体培养基中，37℃，300rpm 震荡培养至OD值0.6左右，加入IPTG至终浓度1mmol/L，降低温度至32℃， 继续培养4小时后离心并收集菌体沉淀。
用缓冲液I(10mmol/L Tris-HCl,1mmol/L EDTA,0.1％Triton X-100, pH8.0)重悬沉淀，300W超声30次(20s超声，20s间隔算一次)，之后17300g 离心20分钟，用缓冲液I洗涤沉淀两次后加入缓冲液II(10mmol/LTris-HCl, 8mol/Lurea,pH8.0)溶解沉淀。
(4)目的蛋白的纯化和复性
目的蛋白的纯化分为两步，首先采用DEAE阴离子交换层析 (ion-exchangechromatography，IEX)，用8m/L尿素平衡层析介质后，将缓 冲液II(10mmol/LTris-HCl,8mol/Lurea,pH8.0)溶解后的上清通过层析柱， 逐级加大洗脱液中NaCl的含量，从而将目的蛋白洗脱，初步与其它大肠杆 菌菌体蛋白分离。之后采用疏水层析(hydrophobicinteraction chromatography，HIC)进行精细纯化，采用Octyl Sepharose 4Fast Flow层析介质，进一步提高目的蛋白的纯度。
目的蛋白的复性在20mmol/LPB(pH8.0)缓冲液中进行，自8m/L尿素开 始逐步降低尿素的浓度，每一浓度下于4℃透析4小时，更换透析液，直至 尿素浓度降为0。
(5)目的蛋白含量和浓度的鉴定
取适量透析之后的目的蛋白，可采用聚丙烯酰胺凝胶电泳(SDS-PAGE) 对目的蛋白的纯度进行鉴定，同时采用Western Blotting(WB)对其抗原特 性进行鉴定。
实施例2
新型冠状病毒RBD基因工程重组蛋白亚单位疫苗与壳聚糖佐剂的混 合，具体方法如下：
将2g壳聚糖放入100mL 1％的食用醋酸中，100℃加热溶解；冷却后作 为佐剂，4℃保存；
将纯化后的新型冠状病毒RBD基因工程重组蛋白亚单位蛋白定容为 0.5mg/mL；
将上述亚单位蛋白与佐剂按照9:1的体积比混合均匀。
每人每次0.4mL，如图1所示喷雾接种。
两次喷雾接种后，分别就血清和鼻腔灌洗液中和抗体进行检测，检测结 果如表1所示。
表1血清和鼻腔灌洗液中和抗体检测结果
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普 通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润 饰，这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 北京尚位生物技术发展有限公司
<120> 一种新型冠状病毒的基因工程重组蛋白、重组载体、重组工程菌和疫苗
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 785
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
ccatggttga gaaaggtatc taccagacct ctaacttccg tgttcagccg accgaatcta 60
tcgttcgttt cccgaacatc accaacctgt gcccgttcgg tgaagtgttc aacgctaccc 120
gtttcgcttc tgtttacgct tggaaccgta aacgtatctc taactgcgtt gctgactact 180
ctgttctgta caactctgct agcttctcta ccttcaaatg ctacggtgtt agtccgacca 240
aactgaacga cctgtgcttc accaacgttt acgctgacag cttcgttatc cgtggtgacg 300
aagttcgtca gatcgctccg ggtcagaccg gtaagatcgc tgactacaac tacaaactgc 360
cggacgactt caccggttgc gttatcgctt ggaactctaa taacctggac tctaaagttg 420
gtggtaacta caactacctg taccgtctgt tccgtaaatc taacctgaaa ccgttcgaac 480
gtgacatctc taccgaaatc taccaggctg gttctactcc gtgcaacggt gttgaaggtt 540
tcaactgcta cttcccgctg cagtcttacg gtttccagcc gaccaacggt gttggttacc 600
agccgtaccg tgttgtggtt ctgagctttg aactgctgca cgctccggct actgtttgcg 660
gtccgaagaa atctaccaac ctggttaaga acaaatgcgt taacttcaac ttcaacggtc 720
tgactggtgg cggtcagtac atcaaagcta actctaaatt catcggtatc ttcgaataac 780
tcgag 785
<210> 2
<211> 241
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val Gln Pro Thr
1 5 10 15
Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly
20 25 30
Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg
35 40 45
Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser
50 55 60
Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu
65 70 75 80
Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg
85 90 95
Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala
100 105 110
Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala
115 120 125
Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr
130 135 140
Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp
145 150 155 160
Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val
165 170 175
Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro
180 185 190
Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe
195 200 205
Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr
210 215 220
Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn Gly Leu Thr
225 230 235 240
Gly
Claims (9)
1.一种新型冠状病毒SARS-CoV-2的基因工程重组蛋白，其特征在于，所述重组蛋白的编码基因如SEQ ID NO.1所示。
2.根据权利要求1所述重组蛋白，其特征在于，所述重组蛋白的氨基酸序列如SEQ IDNO.2所示。
3.一种表达权利要求1或2所述重组蛋白的重组载体，其特征在于，所述重组载体以原核表达载体为基础载体。
4.根据权利要求3所述重组载体，其特征在于，当以pET28a(+)为基础载体时，所述重组蛋白的编码基因插入所述基础载体的NcoI和XhoI酶切位点之间。
5.一种表达权利要求1或2所述重组蛋白的重组工程菌，其特征在于，所述重组工程菌以原核细胞为基础细胞。
6.权利要求1或2所述重组蛋白或利用权利要求5所述重组工程菌生产得到的重组蛋白在制备新型冠状病毒SARS-CoV-2重组亚单位蛋白疫苗中的应用。
7.一种新型冠状病毒SARS-CoV-2重组亚单位蛋白疫苗，其特征在于，包括权利要求1或2所述重组蛋白或利用权利要求5所述重组工程菌生产得到的重组蛋白和佐剂，所述佐剂包括壳聚糖。
8.根据权利要求7所述重组亚单位蛋白疫苗，其特征在于，所述重组蛋白和佐剂的体积比为9:1。
9.权利要求7或8所述重组亚单位蛋白疫苗的制备方法，其特征在于，将壳聚糖溶于食用醋酸中，冷却后作为佐剂；
将权利要求1或2所述重组蛋白或利用权利要求5所述重组工程菌生产得到的重组蛋白稀释至0.5mg/mL，得亚单位蛋白；
将所述亚单位蛋白与佐剂按照9:1的体积比混合，得所述重组亚单位蛋白疫。
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