CN117384953A - 一种过表达基因质粒、构建方法及其用途 - Google Patents
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Abstract
本发明属于基因工程技术领域,公开了一种过表达基因质粒、构建方法及其用途。该构建方法包括:全基因合成Circ_0058514序列,对所获得的基因序列和pcDNA3.1(+)真核表达载体进行限制性内切酶HindIII和KpnI双酶切,并进行连接;将连接产物转化到E.coliTop10感受态细胞,筛选阳性克隆进行扩大培养;抽提过表达circ_0058514基因质粒,酶切后进行测序鉴定。本发明通过过表达Circ_0058514分析巨噬细胞炎症因子表达、吞噬细菌功能的变化,有助于突破现有认知,进一步挖掘Circ_0058514基因功能。
Description
技术领域
本发明属于基因工程技术领域,尤其涉及一种过表达基因质粒、构建方法及其用途。
背景技术
创面感染是下肢开放性骨折最常见的并发症,也是长期困扰骨科领域的难点问题。文献报道,高暴力开放性创伤的患者即使在5小时内接受彻底清创,诊疗期间发生感染的几率仍为28%;开放性骨折GustiloⅢ型严重软组织损伤的患者发生创面及深部感染的风险更高达50%。感染不仅破坏组织的正常结构及功能,而且加重局部缺血、缺氧及过度炎症反应,严重妨碍愈合修复过程,极易引发骨髓炎、骨不连、肢体功能缺失甚至导致截肢,给患者带来巨大痛苦。统计数据表明,创面感染使下肢开放性骨折患者平均住院时间延长54天、平均住院费用增加1.89倍,浪费了大量医疗卫生资源。
巨噬细胞协调下的固有免疫应答在抵御细菌感染与维持免疫稳态中发挥极其重要的作用。巨噬细胞作为组织内的常驻吞噬细胞能在感染发生后迅速识别并吞噬杀菌,是最早做出反应的免疫细胞;同时通过模式识别受体PPRs介导NF-κB、C/EBP、MAPK等信号通路表达炎症因子及趋化因子,不仅促进组织细胞分泌抗菌肽直接杀伤细菌,还使中性粒细胞迅速聚集到创面感染部位清除病原并限制感染播散。然而创伤后机体屏障破坏、血液循环不良及组织损伤等病理变化严重影响巨噬细胞免疫防御功能,迟发性组织坏死所释放的炎性介质进一步损害免疫调节能力。因此,寻找增强巨噬细胞免疫功能的有效治疗手段显得尤为重要。
基因工程,又称为基因重组技术,是在体外对DNA进行操作的一门技术,广泛用于疾病基础研究、基因治疗、基因免疫、基因疫苗等,其三要素分别为:酶、目的基因与载体。而真核表达载体是基因工程中用于构建真核基因表达系统的一种载体。近几十年来,各国学者围绕真核表达载体进行了各种疾病、基因等相关研究,在基因工程方面做了大量工作,为医学科学研究开辟了崭新途径。
环状RNA(circRNA)是一类特殊的非编码RNA分子,与传统的线性RNA(linearRNA)不同,它是通过反向剪接形成的闭合RNA分子。circRNA不具有5'末端帽子和3'末端poly(A)尾巴,通过外显子环化或内含子环化,将3'和5'末端连接起来形成完整的环形结构,避免被核酸外切酶降解,比线性RNA更稳定,更具有保守性。因此circRNA成为新型标记物与生物干预靶点。circ_0058514是一种527nt长的真核非编码环状RNA,可通过竞争性内源RNA(ceRNA)机制在转录及转录后水平对基因表达进行调控,目前认为该基因在肿瘤细胞中发挥调控作用,参与肿瘤的发生发展。研究发现,该基因在巨噬细胞中也有表达。
为了进一步研究circ_0058514的功能,目前急需一种有助于circ_0058514功能研究并且揭示其在感染免疫病程中作用的分子生物学工具。
发明内容
为克服相关技术中存在的问题,本发明公开实施例提供了一种过表达基因质粒、构建方法及其用途。具体涉及一种过表达circ_0058514基因质粒及其构建方法和应用。
所述技术方案如下:一种过表达基因质粒,所述过表达基因质粒的DNA序列为SEQID NO:1。
进一步的,利用所述过表达基因质粒转染制备巨噬细胞,在测定炎性因子表达水平上应用。
进一步的,利用所述过表达基因质粒转染制备增强吞噬细菌能力的巨噬细胞。
本发明的另一目的在于提供一种过表达基因质粒的构建方法,包括以下步骤:
S1,全基因合成Circ_0058514序列,对所获得的基因序列和pcDNA3.1(+)真核表达载体进行限制性内切酶HindIII和KpnI双酶切,并进行连接;
S2,将连接产物转化到E.coliTop10感受态细胞,筛选阳性克隆进行扩大培养;
S3,抽提过表达circ_0058514基因质粒,酶切后进行测序鉴定。
进一步的,在步骤S1具体包括:
Circ_0058514序列和pcDNA3.1(+)载体进行HindIII和KpnI的双酶切反应依照限制性内切酶产品说明书进行,酶切体系如下:HindIII限制性内切酶、KpnI限制性内切酶、10×内切酶buffer、Circ_0058514序列或和pcDNA3.1(+)载体。
进一步的,在步骤S2中,具体包括:
将上述酶切体系放置水浴锅中,并作用;
将上述酶切产物进行琼脂糖凝胶电泳;
回收目的DNA片段;
配制去磷酸化体系:CIAP、10×CIAP缓冲液、pcDNA3.1(+)载体;
配制连接体系:去磷酸化后pcDNA3.1(+)载体、缓冲液、10×T4DNA连接酶、Circ_0058514序列;
将上述反应体系置于离心管中混匀,水浴过夜。
取出储存Top10感受态细胞,冰浴解冻后加入连接产物,混匀,冰上静置;
热休克,取出置冰上;
加入LB培养液,振荡培养;
室温离心后,弃去上清,用剩余上清将菌体沉淀去除,将菌液均匀涂布于预热的LB琼脂平板上,培养箱中倒置培养过夜;
阳性克隆筛选后观察平板上有菌落生长,用无菌吸头挑取单克隆菌落,接种于LB培养液中,置于摇床上振摇培养过夜。
进一步的,在步骤S3中,碱裂解法小量提取重组质粒pcDNA3.1(+)/circ0058514;
酶切后进行测序鉴定包括:配制酶切鉴定体系,混匀后置于水浴;
将酶切产物进行琼脂糖凝胶电泳,鉴定并记录正确的重组质粒;
将质粒进行测序,并与目的片段序列比对,进一步确定质粒构建是否正确;
取正确重组质粒的菌液进行扩增培养,并用质粒小量快速提取试剂盒提取并纯化质粒,保存备用。
本发明的另一目的在于提供一种所述过表达基因质粒在制备用于治疗创面感染下肢开放性骨折药物中的用途。
进一步的,所述过表达基因质粒在制备用于治疗创面感染下肢开放性骨折基因免疫蛋白上的应用。
进一步的,所述过表达基因质粒在制备用于治疗创面感染下肢开放性骨折基因疫苗上的应用。
结合上述的所有技术方案,本发明所具备的优点及积极效果为:本发明的目的在于克服现有技术的不足,提供了一种有助于circ_0058514基因功能分析,并且实现其在巨噬细胞感染免疫病程中过表达的基因质粒及其构建方法和应用。
重组质粒pcDNA3.1(+)/circ_0058514转染入巨噬细胞系RAW264.7中,促进炎症因子的分泌和吞噬细菌。
本发明相比现有技术的优点在于:
运用基因重组技术,针对Circ_0058514基因,构建重组真核表达载体pcDNA3.1(+)/circ_0058514,并将其转染至巨噬细胞系RAW264.7中,为分析Circ_0058514基因功能提供了有用分子生物学工具和构建方法;
通过过表达Circ_0058514分析巨噬细胞炎症因子表达、吞噬细菌功能的变化,有助于突破现有认知,进一步挖掘Circ_0058514基因功能。
附图说明
此处的附图被并入说明书中并构成本说明书的一部分,示出了符合本公开的实施例,并与说明书一起用于解释本公开的原理。
图1是本发明实施例提供的过表达基因质粒的构建方法流程图;
图2是本发明实施例提供的pcDNA3.1(+)/circ_0058514质粒构建模式图;
图3是本发明实施例提供的电泳图;
图4是本发明实施例提供的qRT-PCR确认转染后各组细胞中分子的表达水平图;
图5是本发明实施例提供的pcDNA3.1(+)/circ_0058514促进巨噬细胞炎性因子表达图;
图6是本发明实施例提供的pcDNA3.1(+)/circ_0058514促进巨噬细胞吞噬金葡菌镜像图;
图7是本发明实施例提供图6的柱状图。
具体实施方式
为使本发明的上述目的、特征和优点能够更加明显易懂,下面结合附图对本发明的具体实施方式做详细的说明。在下面的描述中阐述了很多具体细节以便于充分理解本发明。但是本发明能够以很多不同于在此描述的其他方式来实施,本领域技术人员可以在不违背本发明内涵的情况下做类似改进,因此本发明不受下面公开的具体实施的限制。
本发明实施例提供一种过表达基因质粒,所述过表达基因质粒的DNA序列为SEQID NO:1。
利用所述过表达基因质粒转染制备巨噬细胞,在测定炎性因子表达水平上应用。
利用所述过表达基因质粒转染制备增强吞噬细菌能力的巨噬细胞。
实施例1,本发明实施例通过以下技术方案实现的:一种过表达circ_0058514基因质粒,DNA序列为SEQ ID NO:1,由circ_0058514全基因合成序列(DNA序列为SEQ ID NO:2)与pcDNA3.1(+)真核表达载体(DNA序列为SEQ ID NO:3)的重组构建而成;其构成如下。
pcDNA3.1(+)真核表达载体DNA序列为SEQ ID NO:3:
1gacggatcgggagatctcccgatcccctatggtgcactctcagtacaatc
51tgctctgatgccgcatagttaagccagtatctgctccctgcttgtgtgtt
101ggaggtcgctgagtagtgcgcgagcaaaatttaagctacaacaaggcaag
151gcttgaccgacaattgcatgaagaatctgcttagggttaggcgttttgcg
201ctgcttcgcgatgtacgggccagatatacgcgttgacattgattattgac
251tagttattaatagtaatcaattacggggtcattagttcatagcccatata
301tggagttccgcgttacataacttacggtaaatggcccgcctggctgaccg
351cccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagt
401aacgccaatagggactttccattgacgtcaatgggtggagtatttacggt
451aaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccc
501cctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagta
551catgaccttatgggactttcctacttggcagtacatctacgtattagtca
601tcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtgga
651tagcggtttgactcacggggatttccaagtctccaccccattgacgtcaa
701tgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgta
751acaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggag
801gtctatataagcagagctctctggctaactagagaacccactgcttactg
851gcttatcgaaattaatacgactcactatagggagacccaagctggctagc
901gtttaaacttaagcttggtaccgagctcggatccactagtccagtgtggt
951ggaattctgcagatatccagcacagtggcggccgctcgagtctagagggc
1001ccgtttaaacccgctgatcagcctcgactgtgccttctagttgccagcca
1051tctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccac
1101tcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctga
1151gtaggtgtcattctattctggggggtggggtggggcaggacagcaagggg
1201gaggattgggaagacaatagcaggcatgctggggatgcggtgggctctat
1251ggcttctgaggcggaaagaaccagctggggctctagggggtatccccacg
1301cgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagc
1351gtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttctt
1401cccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatc
1451gggggctccctttagggttccgatttagtgctttacggcacctcgacccc
1501aaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgata
1551gacggtttttcgccctttgacgttggagtccacgttctttaatagtggac
1601tcttgttccaaactggaacaacactcaaccctatctcggtctattctttt
1651gatttataagggattttgccgatttcggcctattggttaaaaaatgagct
1701gatttaacaaaaatttaacgcgaattaattctgtggaatgtgtgtcagtt
1751agggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagca
1801tgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctcccca
1851gcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccatagt
1901cccgcccctaactccgcccatcccgcccctaactccgcccagttccgccc
1951attctccgccccatggctgactaattttttttatttatgcagaggccgag
2001gccgcctctgcctctgagctattccagaagtagtgaggaggcttttttgg
2051aggcctaggcttttgcaaaaagctcccgggagcttgtatatccattttcg
2101gatctgatcaagagacaggatgaggatcgtttcgcatgattgaacaagat
2151ggattgcacgcaggttctccggccgcttgggtggagaggctattcggcta
2201tgactgggcacaacagacaatcggctgctctgatgccgccgtgttccggc
2251tgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggt
2301gccctgaatgaactgcaggacgaggcagcgcggctatcgtggctggccac
2351gacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaa
2401gggactggctgctattgggcgaagtgccggggcaggatctcctgtcatct
2451caccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcg
2501gctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaac
2551atcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcag
2601gatgatctggacgaagagcatcaggggctcgcgccagccgaactgttcgc
2651caggctcaaggcgcgcatgcccgacggcgaggatctcgtcgtgacccatg
2701gcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctgga
2751ttcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagc
2801gttggctacccgtgatattgctgaagagcttggcggcgaatgggctgacc
2851gcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgcc
2901ttctatcgccttcttgacgagttcttctgagcgggactctggggttcgaa
2951atgaccgaccaagcgacgcccaacctgccatcacgagatttcgattccac
3001cgccgccttctatgaaaggttgggcttcggaatcgttttccgggacgccg
3051gctggatgatcctccagcgcggggatctcatgctggagttcttcgcccac
3101cccaacttgtttattgcagcttataatggttacaaataaagcaatagcat
3151cacaaatttcacaaataaagcatttttttcactgcattctagttgtggtt
3201tgtccaaactcatcaatgtatcttatcatgtctgtataccgtcgacctct
3251agctagagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattg
3301ttatccgctcacaattccacacaacatacgagccggaagcataaagtgta
3351aagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgc
3401tcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatg
3451aatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccg
3501cttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcg
3551gtatcagctcactcaaaggcggtaatacggttatccacagaatcagggga
3601taacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaacc
3651gtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgac
3701gagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacagg
3751actataaagataccaggcgtttccccctggaagctccctcgtgcgctctc
3801ctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcg
3851ggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggt
3901gtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagc
3951ccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggta
4001agacacgacttatcgccactggcagcagccactggtaacaggattagcag
4051agcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaact
4101acggctacactagaagaacagtatttggtatctgcgctctgctgaagcca
4151gttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccac
4201cgctggtagcggtttttttgtttgcaagcagcagattacgcgcagaaaaa
4251aaggatctcaagaagatcctttgatcttttctacggggtctgacgctcag
4301tggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaag
4351gatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatct
4401aaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagt
4451gaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctg
4501actccccgtcgtgtagataactacgatacgggagggcttaccatctggcc
4551ccagtgctgcaatgataccgcgagacccacgctcaccggctccagattta
4601tcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgc
4651aactttatccgcctccatccagtctattaattgttgccgggaagctagag
4701taagtagttcgccagttaatagtttgcgcaacgttgttgccattgctaca
4751ggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccgg
4801ttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaag
4851cggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgca
4901gtgttatcactcatggttatggcagcactgcataattctcttactgtcat
4951gccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcat
5001tctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaata
5051cgggataataccgcgccacatagcagaactttaaaagtgctcatcattgg
5101aaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagat
5151ccagttcgatgtaacccactcgtgcacccaactgatcttcagcatctttt
5201actttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgc
5251aaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcc
5301tttttcaatattattgaagcatttatcagggttattgtctcatgagcgga
5351tacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcac
5401atttccccgaaaagtgccacctgacgtc。
其中将第917-920的ggta替换为DNA序列为SEQ ID NO:2:“GCGAGGATTAAATCCACCACACAGGGTGAAATCTATCTCCATGACAACATTCACACAACAGGAAATT GAATTCTTACAAAAACATGGAAATGAAGTCTGTAAACAGATTTGGCTAGGATTATTTGATGATAGATCTTCAGCAATTCCAGACTTCAGGGATCCACAAAAAGTGAAAGAGTTTCTACAAGAAAAGTATGAAAAGAAAAGATGGTATGTCCCGCCAGAACAAGCCAAAGTCGTGGCATCAGTTCATGCATCTATTTCAGGGTCCTCTGCCAGTAGCACAAGCAGCACACCTGAGGTCAAACCACTGAAATCTCTTTTAGGGGATTCTGCACCAACACTGCACTTAAATAAGGGCACACCTAGTCAGTCCCCAGTTGTAGGTCGTTCTCAAGGGCAGCAGCAGGAGAAGAAGCAATTTGACCTTTTAAGTGATCTCGGCTCAGACATCTTTGCTGCTCCAGCTCCTCAGTCAACAGCTACAGCCAATTTTGCTAACTTTGCACATTTCAACAGTCATGCAG”。
则构成过表达circ_0058514基因质粒,DNA序列为SEQ ID NO:1:
具体为5951bp“
gacggatcgggagatctcccgatcccctatggtgcactctcagtacaatc
tgctctgatgccgcatagttaagccagtatctgctccctgcttgtgtgtt
ggaggtcgctgagtagtgcgcgagcaaaatttaagctacaacaaggcaag
gcttgaccgacaattgcatgaagaatctgcttagggttaggcgttttgcg
ctgcttcgcgatgtacgggccagatatacgcgttgacattgattattgac
tagttattaatagtaatcaattacggggtcattagttcatagcccatata
tggagttccgcgttacataacttacggtaaatggcccgcctggctgaccg
cccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagt
aacgccaatagggactttccattgacgtcaatgggtggagtatttacggt
aaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccc
cctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagta
catgaccttatgggactttcctacttggcagtacatctacgtattagtca
tcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtgga
tagcggtttgactcacggggatttccaagtctccaccccattgacgtcaa
tgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgta
acaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggag
gtctatataagcagagctctctggctaactagagaacccactgcttactg
gcttatcgaaattaatacgactcactatagggagacccaagctggctagc
gtttaaacttaagcttGCGAGGATTAAATCCACCACACAGGGTGAAATCTATCTCCATGACAACATTCACACAACAGGAAATTGAATTCTTACAAAAACATGGAAATGAAGTCTGTAAACAGATTTGGCTAGGATTATTTGATGATAGATCTTCAGCAATTCCAGACTTCAGGGATCCACAAAAAGTGAAAGAGTTTCTACAAGAAAAGTATGAAAAGAAAAGATGGTATGTCCCGCCAGAACAAGCCAAAGTCGTGGCATCAGTTCATGCATCTATTTCAGGGTCCTCTGCCAGTAGCACAAGCAGCACACCTGAGGTCAAACCACTGAAATCTCTTTTAGGGGATTCTGCACCAACACTGCACTTAAATAAGGGCACACCTAGTCAGTCCCCAGTTGTAGGTCGTTCTCAAGGGCAGCAGCAGGAGAAGAAGCAATTTGACCTTTTAAGTGATCTCGGCTCAGACATCTTTGCTGCTCCAGCTCCTCAGTCAACAGCTACAGCCAATTTTGCTAACTTTGCACATTTCAACAGTCATGCAGccgagc t c
ggatccactagtccagtgtggt
ggaattctgcagatatccagcacagtggcggccgctcgagtctagagggc
ccgtttaaacccgctgatcagcctcgactgtgccttctagttgccagcca
tctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccac
tcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctga
gtaggtgtcattctattctggggggtggggtggggcaggacagcaagggg
gaggattgggaagacaatagcaggcatgctggggatgcggtgggctctat
ggcttctgaggcggaaagaaccagctggggctctagggggtatccccacg
cgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagc
gtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttctt
cccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatc
gggggctccctttagggttccgatttagtgctttacggcacctcgacccc
aaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgata
gacggtttttcgccctttgacgttggagtccacgttctttaatagtggac
tcttgttccaaactggaacaacactcaaccctatctcggtctattctttt
gatttataagggattttgccgatttcggcctattggttaaaaaatgagct
gatttaacaaaaatttaacgcgaattaattctgtggaatgtgtgtcagtt
agggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagca
tgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctcccca
gcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccatagt
cccgcccctaactccgcccatcccgcccctaactccgcccagttccgccc
attctccgccccatggctgactaattttttttatttatgcagaggccgag
gccgcctctgcctctgagctattccagaagtagtgaggaggcttttttgg
aggcctaggcttttgcaaaaagctcccgggagcttgtatatccattttcg
gatctgatcaagagacaggatgaggatcgtttcgcatgattgaacaagat
ggattgcacgcaggttctccggccgcttgggtggagaggctattcggcta
tgactgggcacaacagacaatcggctgctctgatgccgccgtgttccggc
tgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggt
gccctgaatgaactgcaggacgaggcagcgcggctatcgtggctggccac
gacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaa
gggactggctgctattgggcgaagtgccggggcaggatctcctgtcatct
caccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcg
gctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaac
atcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcag
gatgatctggacgaagagcatcaggggctcgcgccagccgaactgttcgc
caggctcaaggcgcgcatgcccgacggcgaggatctcgtcgtgacccatg
gcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctgga
ttcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagc
gttggctacccgtgatattgctgaagagcttggcggcgaatgggctgacc
gcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgcc
ttctatcgccttcttgacgagttcttctgagcgggactctggggttcgaa
atgaccgaccaagcgacgcccaacctgccatcacgagatttcgattccac
cgccgccttctatgaaaggttgggcttcggaatcgttttccgggacgccg
gctggatgatcctccagcgcggggatctcatgctggagttcttcgcccac
cccaacttgtttattgcagcttataatggttacaaataaagcaatagcat
cacaaatttcacaaataaagcatttttttcactgcattctagttgtggtt
tgtccaaactcatcaatgtatcttatcatgtctgtataccgtcgacctct
agctagagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattg
ttatccgctcacaattccacacaacatacgagccggaagcataaagtgta
aagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgc
tcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatg
aatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccg
cttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcg
gtatcagctcactcaaaggcggtaatacggttatccacagaatcagggga
taacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaacc
gtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgac
gagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacagg
actataaagataccaggcgtttccccctggaagctccctcgtgcgctctc
ctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcg
ggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggt
gtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagc
ccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggta
agacacgacttatcgccactggcagcagccactggtaacaggattagcag
agcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaact
acggctacactagaagaacagtatttggtatctgcgctctgctgaagcca
gttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccac
cgctggtagcggtttttttgtttgcaagcagcagattacgcgcagaaaaa
aaggatctcaagaagatcctttgatcttttctacggggtctgacgctcag
tggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaag
gatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatct
aaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagt
gaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctg
actccccgtcgtgtagataactacgatacgggagggcttaccatctggcc
ccagtgctgcaatgataccgcgagacccacgctcaccggctccagattta
tcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgc
aactttatccgcctccatccagtctattaattgttgccgggaagctagag
taagtagttcgccagttaatagtttgcgcaacgttgttgccattgctaca
ggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccgg
ttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaag
cggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgca
gtgttatcactcatggttatggcagcactgcataattctcttactgtcat
gccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcat
tctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaata
cgggataataccgcgccacatagcagaactttaaaagtgctcatcattgg
aaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagat
ccagttcgatgtaacccactcgtgcacccaactgatcttcagcatctttt
actttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgc
aaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcc
tttttcaatattattgaagcatttatcagggttattgtctcatgagcgga
tacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcac
atttccccgaaaagtgccacctgacgtc。
如图1所示,本发明实施例提供的过表达circ_0058514基因质粒的构建方法包括:
S1,全基因合成Circ_0058514序列(由上海生工生物工程股份有限公司合成),对所获得的基因序列和pcDNA3.1(+)真核表达载体进行限制性内切酶HindIII和KpnI双酶切,并进行连接;
S2,将连接产物转化到E.coliTop10感受态细胞,筛选阳性克隆进行扩大培养;
S3,抽提过表达circ_0058514基因质粒,酶切后进行测序鉴定。
通过上述实施例,本发明可应用于感染控制领域,针对感染进行早期预防及控制,及时建立有效的免疫防御。
环状RNA能在基因水平对细胞功能进行调控,不仅结构稳定且序列高度保守,是精准化的免疫调控技术。
局部免疫受损是创面感染发生、发展及迁延不愈的重要原因。本发明从临床诊疗实践出发,针对聚焦清创后如何及时建立有效免疫防御的技术难题,提出通过circ0058514调控巨噬细胞功能进而增强创面免疫防御的新构想,可解决现有技术难题。
实施例2,如图2所示,本发明实施例提供的过表达circ_0058514基因质粒的构建方法具体包括如下步骤:
步骤1,Circ_0058514序列和pcDNA3.1(+)载体进行HindIII和KpnI的双酶切反应依照限制性内切酶产品说明书进行,酶切体系如下:HindIII限制性内切酶、KpnI限制性内切酶、10×内切酶buffer、Circ_0058514序列或和pcDNA3.1(+)载体;
载体双酶切体系
步骤2,将上述反应体系放置37℃水浴锅中,并作用4h;
步骤3,将上述酶切产物进行1%琼脂糖凝胶电泳;
步骤4,观察结果并回收目的DNA片段。
步骤5,配制去磷酸化体系:CIAP1μl、10×CIAP缓冲液1μl、pcDNA3.1(+)载体8μl
步骤6,配制连接体系:去磷酸化后pcDNA3.1(+)载体0.5μl、缓冲液1μl、10×T4DNA连接酶1μl、Circ_0058514序列7.5μl;
步骤7,将上述反应体系置于0.5ml的离心管中混匀,16℃水浴过夜。
步骤8,取出储存于﹣80℃的Top10感受态细胞,冰浴解冻后加入10μl连接产物,混匀,冰上静置30min;
步骤9,42℃热休克90s,迅速取出置冰上3min;
步骤10,加入500μlLB培养液,37℃、250rpm振荡培养45min;
步骤11,室温7000rpm离心1min后,弃去约450μL上清,用约100μL剩余上清将菌体沉淀轻轻吹起后,将菌液均匀涂布于37℃预热的LB琼脂平板(含氨苄青霉素100μg/mL)上,于37℃培养箱中倒置培养过夜。
步骤12,阳性克隆筛选约16-18h后观察平板上有白色菌落生长,用无菌吸头挑取10个单克隆菌落,接种于2mLLB培养液(含氨苄青霉素100μg/mL)中,置于摇床上37℃220rpm振摇培养过夜。
步骤13,碱裂解法小量提取重组质粒pcDNA3.1(+)/circ0058514,包括:
(a)取1ml过夜培养的菌液转入无菌1.5ml离心管中,以7000rmp,1min离心后,倒扣在吸水纸上彻底弃上清。
(b)加入100μLBufferI(含100μg/mLRNaseA,4℃贮存),充分涡旋重悬细菌。
(c)加入200μLBufferII(当日配制),立即计时3min并加盖轻柔翻转混匀3~4次。
(d)加入150μLBufferIII(4℃贮存),轻柔翻转混匀后冰置10min。
(e)4℃,12000rmp离心10min,将上清吸至新的无菌1.5mL离心管中。
(f)加入1mL无水乙醇(-20℃预冷),充分混匀后冰置10min。
(g)4℃,12000rmp离心10min,小心弃去上清,加1mL75%乙醇洗涤DNA沉淀。
(h)室温,12000rmp离心5min,小心弃尽上清。
(i)室温干10min,至沉淀呈现半透明状。
(j)用20μL1×TE(PH8.0,含20μg/mLRNaseA)溶解DNA沉淀,室温放置20min,使DNA充分溶解,置4℃近期备用,或置-20℃长期保存。
步骤14,重组质粒酶切鉴定:
配制酶切鉴定体系
混匀后置于37℃水浴4h。
步骤15,将酶切产物进行1.5%琼脂糖凝胶电泳,鉴定并记录正确的重组质粒(载体质粒含5.4Kb和527bp的片段条带)。电泳图如图3所示。
步骤16,将质粒进行测序,并与目的片段序列比对,进一步确定质粒构建是否正确。
步骤17,取正确重组质粒的菌液进行扩增培养,并用质粒小量快速提取试剂盒提取并纯化质粒,-20℃保存备用。
circ_0058514的SEQ ID NO:2的DNA基因测序结果如下:GCGAGGATTAAATCCACCACACAGGGTGAAATCTATCTCCATGACAACATTCACACAACAGGAAATTGAATTCTTACAAAAACATGGAAATGAAGTCTGTAAACAGATTTGGCTAGGATTATTTGATGATAGATCTTCAGCAATTCCAGACTTCAGGGATCCACAAAAAGTGAAAGAGTTTCTACAAGAAAAGTATGAAAAGAAAAGATGGTATGTCCCGCCAGAACAAGCCAAAGTCGTGGCATCAGTTCATGCATCTATTTCAGGGTCCTCTGCCAGTAGCACAAGCAGCACACCTGAGGTCAAACCACTGAAATCTCTTTTAGGGGATTCTGCACCAACACTGCACTTAAATAAGGGCACACCTAGTCAGTCCCCAGTTGTAGGTCGTTCTCAAGGGCAGCAGCAGGAGAAGAAGCAATTTGACCTTTTAAGTGATCTCGGCTCAGACATCTTTGCTGCTCCAGCTCCTCAGTCAACAGCTACAGCCAATTTTGCTAACTTTGCACATTTCAACAGTCATGCAG。
实施例3,下面结合具体应用对本发明技术方案作进一步描述。
应用一、重组质粒转染巨噬细胞测定炎性因子表达。
1、细胞准备:
(1.1)对数生长期的RAW264.7细胞接种于12孔培养瓶中,将细胞瓶在37℃,5%CO2培养箱中预培养18-24h。
质粒转染:
在无菌的1.5mL离心管中配制A、B转染液(以下为每孔细胞所用转染液量)。
A液:用无血清培养基Opti-MEM分别稀释各质粒各1.5μg,终量150μL,轻轻混匀。
B液:用无血清培养基Opti-MEM稀释1.5μL脂质体Lipofectamine2000Reagent,终量150μL,轻轻混匀,室温静置5min。将B液缓慢滴加入A液中轻轻混合,室温静置20min。
(1.2)PBS洗细胞一次,将上述混合转染液加入每孔中,300μL/孔,轻晃培养板使其分布均匀后,置于37℃、5%CO2的细胞培养箱中培养,4-6h后更换为正常培养基继续培养48h,qRT-PCR确认转染后各组细胞中circ0058514的表达水平。
(1.3)细胞分组:
Control;
pcDNA3.1;
pcDNA3.1/circ0058514;
按照上述分组对细胞进行转染,收集培养液上清;将各组细胞与金葡菌进行共培养后,收集培养液上清进行ELISA检测。
2、ELISA检测细胞上清中TNF-α、IL-1β、IL-8、CXCL1的表达。
(i)将酶联免疫分析试剂盒从4℃冰箱中取出,室温平衡30min。
(ii)标准品加样:设置标准品孔和样品孔,标准品孔各加不同浓度的标准品50μL。
(iii)加样:分别设空白孔(空白对照孔不加样品及酶标试剂,其余各步操作相同)、待测样品孔。在酶标包被板上待测样品孔中加待测样品50μL,加于酶标板孔底部,尽量不触及孔壁,轻轻晃动混匀。
(iv)温育:用封板膜封板后置37℃温育30min。
(v)配液:将20倍浓缩洗涤液用蒸馏水20倍稀释后备用。
(vi)洗涤:小心揭掉封板膜,弃去液体,甩干,每孔加满洗涤液,静置30s后弃去,如此重复5次,拍干。加酶:每孔加入酶标试剂50μL,空白孔除外。
(vii)温育:操作同步骤(iv)。
(viii)洗涤:操作同(vi)。
(iX)显色:每孔先加入显色剂A50μL,再加入显色剂B50μL,轻轻震荡混匀,37℃避光显色15min。
(Xi)终止:每孔加终止液50μL,终止反应(此时蓝色立转黄色)。
(Xii)测定:以空白空调零,450nm波长依序测量各孔的吸光度(OD值)。测定应在加终止液后15min以内进行。其中,qRT-PCR确认转染后各组细胞中分子的表达水平如图4所示,pcDNA3.1(+)/circ_0058514促进巨噬细胞炎性因子表达如图5所示。
应用二、共聚焦观察巨噬细胞吞噬细菌试验。
细胞准备:
将对数生长期的RAW264.7细胞接种于含有玻片的24孔培养板中,将细胞置于37℃,5%CO2培养箱中预培养18-24h。
质粒转染:
在无菌的1.5mL离心管中配制A、B转染液(以下为每孔细胞所用转染液量)。
A液:用无血清培养基Opti-MEM分别稀释各质粒各1.2μg,终量100μL,轻轻混匀。
B液:用无血清培养基Opti-MEM稀释1.2μL脂质体Lipofectamine2000Reagent,终量100μL,轻轻混匀,室温静置5min。将B液缓慢滴加入A液中轻轻混合,室温静置20min。
PBS洗细胞一次,将上述混合转染液加入每孔中,1000μL/孔,轻晃培养板使其分布均匀后,置于37℃、5%CO2的细胞培养箱中培养,4-6h后更换为正常培养基继续培养48h。
细胞分组:
Control;
PcDNA3.1/circ0058514。
吞噬试验及共聚焦拍照:向转染后的细胞加入FITC-d-Lys标记的金黄色葡萄球菌,进行细菌细胞共培养。
向每孔中加入300μL4%多聚甲醛固定细胞,室温静置30min。
吸去液体,用预冷的PBS洗细胞三次,5min/次。
吸去PBS,向每孔中加入300μL0.05%Triton-X-100(PBS稀释)进行通透处理,4℃作用5min。
吸去液体,用预冷的PBS洗细胞三次,5min/次。
向每孔中加入300μLDAPI(1:1000稀释,终浓度1μg/ml),4℃作用5min。
吸去液体,用预冷的PBS洗细胞三次,5min/次。
向载玻片上滴加3μL荧光保护剂,取出孔板中的玻片,倒置扣于荧光保护剂上。
共聚焦显微镜下观察荧光并以细胞核为内参计量吞噬金葡菌荧光强度。
其中,pcDNA3.1(+)/circ_0058514促进巨噬细胞吞噬金葡菌镜像图如图6所示,柱状图如图7所示。
在上述实施例中,对各个实施例的描述都各有侧重,某个实施例中没有详述或记载的部分,可以参见其它实施例的相关描述。
以上所述,仅为本发明较优的具体的实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,都应涵盖在本发明的保护范围之内。
Claims (10)
1.一种过表达基因质粒,其特征在于,所述过表达基因质粒的DNA序列为SEQ ID NO:1。
2.根据权利要求1所述过表达基因质粒,其特征在于,利用所述过表达基因质粒转染制备巨噬细胞,在测定炎性因子表达水平上应用。
3.根据权利要求1所述过表达基因质粒,其特征在于,利用所述过表达基因质粒转染制备增强吞噬细菌能力的巨噬细胞。
4.一种过表达基因质粒的构建方法,其特征在于,该构建方法包括以下步骤:
S1,全基因合成Circ_0058514序列,对所获得的基因序列和pcDNA3.1(+)真核表达载体进行限制性内切酶HindIII和KpnI双酶切,并进行连接;
S2,将连接产物转化到E.coliTop10感受态细胞,筛选阳性克隆进行扩大培养;
S3,抽提过表达circ_0058514基因质粒,酶切后进行测序鉴定。
5.根据权利要求4所述的过表达基因质粒的构建方法,其特征在于,在步骤S1具体包括:
Circ_0058514序列和pcDNA3.1(+)载体进行HindIII和KpnI的双酶切反应依照限制性内切酶产品说明书进行,酶切体系如下:HindIII限制性内切酶、KpnI限制性内切酶、10×内切酶buffer、Circ_0058514序列或和pcDNA3.1(+)载体。
6.根据权利要求4所述的过表达基因质粒的构建方法,其特征在于,在步骤S2中,具体包括:
将上述酶切体系放置水浴锅中,并作用;
将上述酶切产物进行琼脂糖凝胶电泳;
回收目的DNA片段;
配制去磷酸化体系:CIAP、10×CIAP缓冲液、pcDNA3.1(+)载体;
配制连接体系:去磷酸化后pcDNA3.1(+)载体、缓冲液、10×T4DNA连接酶、Circ_0058514序列;
将上述反应体系置于离心管中混匀,水浴过夜;
取出储存Top10感受态细胞,冰浴解冻后加入连接产物,混匀,冰上静置;
热休克,取出置冰上;
加入LB培养液,振荡培养;
室温离心后,弃去上清,用剩余上清将菌体沉淀去除,将菌液均匀涂布于预热的LB琼脂平板上,培养箱中倒置培养过夜;
阳性克隆筛选后观察平板上有菌落生长,用无菌吸头挑取单克隆菌落,接种于LB培养液中,置于摇床上振摇培养过夜。
7.根据权利要求4所述的过表达基因质粒的构建方法,其特征在于,在步骤S3中,碱裂解法小量提取重组质粒pcDNA3.1(+)/circ0058514;
酶切后进行测序鉴定包括:配制酶切鉴定体系,混匀后置于水浴;
将酶切产物进行琼脂糖凝胶电泳,鉴定并记录正确的重组质粒;
将质粒进行测序,并与目的片段序列比对,进一步确定质粒构建是否正确;
取正确重组质粒的菌液进行扩增培养,并用质粒小量快速提取试剂盒提取并纯化质粒,保存备用。
8.一种如权利要求1所述过表达基因质粒在制备用于治疗创面感染下肢开放性骨折药物中的用途。
9.根据权利要求8所述的用途,其特征在于,所述过表达基因质粒在制备用于治疗创面感染下肢开放性骨折基因免疫蛋白上的应用。
10.根据权利要求8所述的用途,其特征在于,所述过表达基因质粒在制备用于治疗创面感染下肢开放性骨折基因疫苗上的应用。
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