CN116555346B - 一种采用碳纳米管基因载体递送***促进草鱼生长的方法 - Google Patents
一种采用碳纳米管基因载体递送***促进草鱼生长的方法 Download PDFInfo
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Abstract
本发明公开了一种采用碳纳米管基因载体递送***促进草鱼生长的方法,包括以下步骤:构建草鱼生长激素基因重组质粒;对碳纳米管进行功能化修饰;用超声波将功能化修饰碳纳米管分散于水中,将功能化修饰碳纳米管与水体中草鱼生长激素基因重组质粒结合,得碳纳米管基因载体递送***;将碳纳米管基因载体递送***溶于水中,将草鱼在碳纳米管基因载体递送***水溶液中浸泡,通过浸泡的方式将碳纳米管基因载体递送***进入草鱼细胞,使草鱼生长激素基因在草鱼细胞内进行过表达,从而促进草鱼的生长。还公开了上述碳纳米管基因载体递送***在制备促进草鱼生长产品中的应用。
Description
技术领域
本发明属于水产生物技术领域,具体涉及一种采用碳纳米管基因载体递送***促进草鱼生长的方法。
背景技术
碳纳米管(Carbon Nanotubes,CNTs)是一种无缝、中空的管状物质,由单层石墨片卷曲形成的为单壁碳纳米管(SWCNTs),由多层石墨片卷曲形成的即为多壁碳纳米管(MWCNTs)。碳纳米管具有十分优越的性能,比如大的比表面积以及吸附性能,它们可以非共价和共价结合药物,生物分子以及纳米粒子,独特的管状结构或者说针状结构使其具有较高的穿透能力,可以轻松穿透细胞膜等结构,增强将多肽、蛋白质和核酸输送到细胞内的能力,极大的比表面积有利于与更多的核酸结合,大大提高了负载量。很多研究显示碳纳米管经过功能化修饰后可以显著降低其生物毒性,使其具有更好的生物相容性。
碳纳米管本身不溶于水,在水中的分散性很差,但通过功能化修饰、超声波等化学、物理方法相结合可以大大提高碳纳米管在水中的分散性以及更好地连接基因载体。同时鱼类本身就拥有很大的粘膜表面(皮肤、鳃、肠道和鼻腔粘膜等),因此通过浸泡的方式向鱼体细胞递送质粒不仅大大缩短递送时间,也更容易实现大规模化,更经济适用。
草鱼(Ctenopharyngodon idella)是鲤形目,鲤科,雅罗鱼亚科,“四大家鱼”之一。草鱼是我国重要的经济鱼类,是淡水养殖产量最大的鱼类,提升草鱼品质,促进草鱼生长对于促进草鱼养殖业的可持续健康发展有着重要的意义。生长激素(Growth hormone,GH)是包括鱼类在内的所有脊椎动物脑下垂体产生的一种单链多肽激素,在鱼类中,参与鱼的生长代谢,促进体细胞生长;另外,还具有调节渗透压、电解质平衡等与新陈代谢有关的功能,在鱼类水产养殖业上具有重大的作用。克隆编码生长激素的生长激素基因连接表达载体转入鱼体细胞进行过表达是促进鱼类生长的一个常用方法,传统的基因转运方法:细菌转化、病毒转染、显微注射等都存在一定的局限性,且不易实现规模化操作,而将功能化修饰后的碳纳米管作为基因转运载体具有携载率高、穿膜能力强、成本低、细胞毒性小等优点,因此,利用碳纳米管为载体携带草鱼生长激素基因过表达载体进入草鱼细胞具有很大的可行性来达到促生长的目的。
本发明拟借助碳纳米管这一载体向草鱼细胞递送基因载体,实现生长激素基因在鱼体内的过表达,促进草鱼的生长,形成一种基于碳纳米管基因载体递送***促进草鱼生长的方法,并进一步将碳纳米管基因载体递送***制备成能促进草鱼生长的产品。
发明内容
本发明的目的在于提供一种采用碳纳米管基因载体递送***促进草鱼生长的方法。
本发明的目的还在于提供碳纳米管基因载体递送***在制备促进草鱼生长产品中的应用。
本发明的上述第一个目的可以通过以下技术方案来实现:一种采用碳纳米管基因载体递送***促进草鱼生长的方法,包括以下步骤:
(S1)构建草鱼生长激素基因重组质粒;
(S2)对碳纳米管进行功能化修饰;
(S3)用超声波将功能化修饰碳纳米管分散于水中,将功能化修饰碳纳米管与水体中草鱼生长激素基因重组质粒结合,得碳纳米管基因载体递送***;
(S4)将碳纳米管基因载体递送***溶于水中,将草鱼在碳纳米管基因载体递送***水溶液中浸泡,通过浸泡的方式将碳纳米管基因载体递送***进入草鱼细胞,使草鱼生长激素基因在草鱼细胞内进行过表达,从而促进草鱼的生长。
本发明方法首先构建草鱼生长激素基因重组质粒,对碳纳米管进行功能化修饰使其很好地连接过表达载体并增加在水中的分散性,用超声波将功能化修饰后的碳纳米管分散于水中,与水体中草鱼生长激素基因重组质粒结合,通过浸泡的方式碳纳米管携带重组质粒进入草鱼细胞,生长基因在细胞内进行过表达,从而促进草鱼的生长。
在该采用碳纳米管基因载体递送***促进草鱼生长的方法中:
可选地,步骤(S1)中构建草鱼生长激素基因重组质粒包括:取草鱼垂体组织样品进行总RNA的提取,反转录得到cDNA,设计引物克隆得到目的基因草鱼GH基因,将pcDNA4.0质粒双酶切后与目的基因草鱼GH基因进行连接得到目的基因重组质粒pcDNA4.0-草鱼GH基因,其中所述引物包括上游引物GH-F和下游引物GH-R,所述上游引物GH-F的序列如SEQ IDNO:1所示,下游引物GH-R的序列如SEQ ID NO:2所示。
可选地,步骤(S2)中对碳纳米管进行功能化修饰包括:用浓硫酸与浓硝酸的混合酸将碳纳米管进行氧化,使其增加官能团羧基,随后与大分子物质聚乙烯亚胺PEI进行氨化反应结合,所得产物经洗涤后冻干得到功能化修饰的碳纳米管。
可选地,所述浓硫酸与浓硝酸的体积比为2:1~4:1,更佳为3:1,氧化时间为45~50h,更佳为48 h,所述聚乙烯亚胺PEI的分子量为900~1800 Da,更佳为1800 Da,氨化时间为70~75 h,更佳为72 h,所述碳纳米管为单壁碳纳米管,所述单壁碳纳米管的规格为:OD值:1~2 nm;长度:1~3 μm;纯度:>90 %。
可选地,步骤(S3)中用超声波将功能化修饰碳纳米管分散于水中的时间为30~60min,更佳为40 min。
可选地,步骤(S3)中所述功能化修饰碳纳米管与草鱼生长激素基因重组质粒的质量份配比为2~8:1,更佳为6:1。
可选地,步骤(S4)中所述碳纳米管基因载体递送***水溶液的浓度为1~10 mg/L,更佳为5 mg/L。
可选地,步骤(S4)中将草鱼在碳纳米管基因载体递送***水溶液中浸泡6~12 h,更佳为8 h。
因此,作为本发明的一种优选的实施方式,本发明提供的一种采用碳纳米管基因载体递送***促进草鱼生长的方法,包括以下步骤:
(S1)基因的克隆及重组:取草鱼垂体组织样品进行总RNA的提取,反转录得到cDNA,设计引物克隆得到目的基因,将质粒双酶切后与目的基因进行连接得到目的基因重组质粒,测序验证成功后扩培得到大量的重组质粒;
(S2)碳纳米管的功能化修饰:用浓硫酸与浓硝酸体积比为3:1的混合酸将碳纳米管进行氧化,使其增加官能团羧基(-COOH),随后进行氨化,与大分子物质聚乙烯亚胺(PEI)反应结合,纯水洗涤之后冻干得到功能化修饰的碳纳米管,氨化后的碳纳米管在水中分散性提高,可以吸附质粒并携带其进入鱼体细胞;
(S3)碳纳米管与重组质粒的连接:将碳纳米管用超声波均匀分散在水中40 min,设置一系列碳纳米管与质粒不同质量比(2:1~8:1)进行连接,电泳跑琼脂糖凝胶来鉴定其连接效果,选择合适的质量比例6:1用于后续的实验;
(S4)浸泡处理:将生长激素基因重组质粒与功能化修饰后的单壁碳纳米管连接成功的产物稀释到水体至5 mg/L,使草鱼在该水体中浸泡8 h,碳纳米管浸泡之后转入正常水体饲养。
本发明的上述第二个目的可以通过以下技术方案来实现:碳纳米管基因载体递送***在制备促进草鱼生长产品中的应用。
可选地,所述碳纳米管基因载体递送***主要由功能化修饰碳纳米管和草鱼生长激素基因重组质粒结合而成,所述草鱼生长激素基因重组质粒为pcDNA4.0-草鱼GH基因。
与现有技术相比,本发明具有以下优点:
(1)本发明提供了一种基因载体递送方式来实现生长基因在草鱼内的过表达,促进了草鱼的生长,为制备促进鱼类生长的产品提供了新思路;
(2)本发明利用功能化修饰后的碳纳米管携带草鱼生长激素基因重组质粒通过浸泡的方式进入草鱼细胞,对鱼体内基因进行调控;
(3)本发明通过浸泡的方式向鱼体细胞递送生长激素基因过表达载体可以实现大批量操作,有利于实现规模化,省时省力;
(4)本发明浸泡的方式会大大减少鱼体的伤害,减少实验对鱼带来的副作用,提高鱼苗的成活率;
(5)本发明碳纳米管基因载体递送手段适用于不同生长阶段的鱼体,可以在不同时期实现生长激素基因的过表达。
附图说明
图1是实施例1中草鱼生长激素基因重组质粒构建完成后进行菌落验证的凝胶电泳图;其中1为Marker、2-4为3个平行重复;
图2是实施例1中功能化修饰碳纳米管与草鱼生长激素基因重组质粒的不同质量配比的凝胶电泳图;其中1为质粒对照、2-5分别为碳纳米管与质粒质量比为2:1、4:1、6:1、8:1;
图3是实施例1中草鱼浸泡处理3天后脑、肌肉、性腺三种组织中各个处理组的GH基因表达水平差异图;
图4是实施例1中实验前后草鱼各个处理组生长差异图。
具体实施方式
以下实施例仅用于阐述本发明,而本发明的保护范围并非仅仅局限于以下实施例。所述技术领域的普通技术人员依据以上本发明公开的内容和各参数所取范围,均可实现本发明的目的。以下具体实施例中所用试剂或材料,如未特别说明,均来源于商业渠道。
实施例1
本发明提供的采用碳纳米管基因载体递送***促进草鱼生长的方法,包括以下步骤:
1、草鱼生长基因的克隆及重组:
用新鲜草鱼取垂体样本,进行总RNA的提取并反转录得到cDNA,根据 GenBank(https://www.ncbi.nlm.nih.gov/genbank/)报道的草鱼生长激素 cDNA 用prime blast(https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome)设计克隆引物,由北京擎科生物科技有限公司进行合成,进行PCR反应得到草鱼GH基因(序列如SEQ ID NO:1所示,见下表1)。
PCR反应体系包括:2xTaq Plus Master Mix II (Vazyme, China) 25 μL,GH-F(序列如SEQ ID NO:2所示,下表1)和GH-R(序列如SEQ ID NO:3所示,下表1)引物各2.5 μL,DNA模板2.5 μL,ddH2O 17.5 μL。
PCR扩增反应条件:先94 ℃5 min; 94 ℃15 s, 60 ℃退火20 s,72 ℃延伸40 s,共35个循环; 最后72 ℃延伸10 min。
对pcDNA4.0进行扩培并提取,主要操作步骤包括:取 1 支 100 µL 感受态于冰上解冻 30 min,分为 3 管;每管加入 3 µL 质粒,再冰浴 30 min;42 ℃热激 45 s,不要搅动,立即冰浴;加入 1000 µL 无抗的 LB 液体培养基,180 rpm 震荡 37 ℃培养 45 min;将菌液全部吸入 40 mL 氨苄青霉素的 LB 液体培养基,180rpm 震荡 37 ℃培养过夜;使用 Omega 牌 Endo-free Plasmid Mini Kit II(无内毒素小量质粒提取试剂盒 II)进行提取。
结合 pcDNA4.0 载体和目的基因序列,选择 BamHI 和 EcoRI 两个酶切位点,使用天根生化科技(北京)有限公司无缝克隆引物设计工具设计同源重组引物,由广州擎科生物科技有限公司合成,分别为:GH-F1(序列如SEQ ID NO:4所示,下表1),GH-R1:(序列如SEQID NO:5所示,下表1),进行PCR反应后用 1%琼脂糖凝胶中电泳检测扩增产物结果,用Omega 牌 Gel Extraction Kit(胶回收试剂盒)进行胶回收。选择BamH I 和 EcoR I两个限制性内切酶对质粒进行双酶切,与目的基因连接后进行转化,筛选阳性克隆进行菌落验证,如图1所示,三个平行重复均有明显条带,为阳性,将 PCR 初步鉴定为阳性的菌液送至广州擎科生物公司测序以保证结果的准确。得到目的基因重组质粒,扩培得到更多的重组质粒。
表1 草鱼GH基因及引物序列
SEQ ID | 序列 |
NO:1 | ATGGCTAGAGCATTAGTGCTGTTGTCGGTGGTGCTGGTTAGTTTGTTGGTGAACCAGGGGACGGCCTCAGAGAACCAGCGGCTCTTCAACAACGCAGTCATACGTGTTCAACACCTGCACCAGCTGGCTGCAAAAATGATTAACGACTTTGAGGACAACCTGTTGCCTGAGGAACGCAGACAGCTGAGTAAAATCTTTCCTCTGTCTTTCTGCAACTGTGACTCAATTGAGGCGCCCACTGGAAAAGATGAAACGCAGAAGAGCTCTATGTTGAAGCTCCTTCGCATCTCTTTCCGCCTCATTGAGTCCTGGGAGTTCCCCAGCCAGACCCTGAGCGGCCAGGTCTCAAACAGTCTGACCGTCGGGAACCCCAACCAGATCACTGAGAAGCTGGCCGACTTGAAAGTGGGCATCAGCGTGCTCATCAAGGGATGTCTGGATGGTCAACCAAACATGGATGATAACGACTCCCTGCCACTGCCTTTTGAGGATTTCTACTTGACCATGGGGGAGAGCAGCCTCAGAGAGAGCTTTCGTCTTCTGGCTTGCTTCAAGAAGGACATGCACAAGGTGGAAACTTACCTGAGGGTTGCGAATTGCAGGAGATCCCTGGATTCAAACTGCACCCTGTAG |
NO:2 | ATGGCTAGAGCATTAGTGCTGTTG |
NO:3 | CTACAGGGTGCAGTTTGAATCCAG |
NO:4 | TTGGTACCGAGCTCGGATCCATGGCTAGAGCATTAGTGCTGT |
NO:5 | TCTGGATATCTGCAGAATTCCTACAGGGTGCAGTTTGAATCC |
NO:6 | CCTTCTTGGGTATGGAATCTTG |
NO:7 | AGAGTATTTACGCTCAGGTGGG |
NO:8 | TTGTCGGTGGTGCTGGTTAG |
NO:9 | CACAGTTGCAGAAAGACAGAGG |
2、碳纳米管的功能化修饰
从成都中科时代纳能科技有限公司购得碳纳米管(TNSS,OD:1-2 nm;Length:1-3µm;Purity:>90%);
碳纳米管用浓硝酸和浓硫酸以1:3的体积比混合后酸化48小时左右,将酸稀释后用真空抽滤泵除酸,并透析碳纳米管进行洗涤;
其中浓硫酸或浓硝酸为一般市售浓硫酸浓硝酸,浓硫酸一般是指质量分数大于或等于70%的硫酸溶液,浓硝酸一般是指质量分数约为68%的硝酸溶液。
配0.1 mol/L的吗啉乙磺酸(MES)缓冲液,超声15 min将30 mg碳纳米管均匀分散在20 mL的吗啉乙磺酸(MES)缓冲液中;
加入120 mg的N-羟基琥珀酰亚胺(NHS)与90 mg的1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐(EDC),反应2小时;离心,并使用纯水洗涤沉淀;
将沉淀分散在20 mL的N,N-二甲基甲酰胺溶剂(DMF)中,加入PEI(聚乙烯亚胺,分子量1800 Da),将混合物超声10分钟,分散液在50°C搅拌3天,然后离心用纯水洗涤除去多余的PEI;
配置pH = 1的盐酸水溶液,将沉淀放入20 mL的盐酸溶液中。离心用纯水洗涤两次;
重新将沉淀分散与纯水中,超声40 min后,按碳纳米管与质粒质量比为2:1、4:1、6:1、8:1的比例混合碳纳米管与质粒,吹打混匀;琼脂糖凝胶鉴定并选择合适的比例进行后续实验。
如图2所示,五条条带分别为:质粒对照,碳纳米管与质粒质量比为2:1、4:1、6:1、8:1的连接组,随着碳纳米管与质粒质量比的增大,条带亮度随之减弱,证明碳纳米管可以有效结合质粒。质量比在6:1的条件下条带明显弱于2:1和4:1的条件,与8:1的条带相似,因此选择合适的质量比为6:1。
3、浸泡处理
将修饰后的碳纳米管分散于水中,用超声波进行均匀分散,使其浓度为6 mg/mL。
将目的基因重组质粒溶于水中,配为使其浓度为1 mg/mL。
将碳纳米管与质粒混合,混合均匀后,得到碳纳米管基因载体递送***SWCNTs(单壁碳纳米管)-pcDNA-GH产物。
将上述制备的SWCNTs -pcDNA-GH用水逐级稀释,分别得到重组质粒浓度为1、5、10mg/L的体系,分别为实验组1(1 mg/L),实验组2(5 mg/L),实验组3(10 mg/L),同时设置两个对照组:空白对照组、SWCNTs对照组(与实验组3碳纳米管浓度相同),每组60条鱼。对鱼进行浸浴处理8 h后,将鱼转入正常水体中进行饲喂。
4、GH基因过表达检测
在浸泡处理后三天后从各个处理组随机挑选3条草鱼,取脑、肌肉、性腺组织样品,用Trizol法进行总RNA提取,使用ReverTra Ace qPCR RT Master Mix with gDNA Remove试剂盒将RNA样品反转录为cDNA,设计并合成草鱼β-actin基因及GH基因特异性引物,分别为:β-actin-F(序列如SEQ ID NO:6所示,上表1)、β-actin-R(序列如SEQ ID NO:7所示,上表1)、GH-F2(序列如SEQ ID NO:8所示,上表1)、GH-R2(序列如SEQ ID NO:9所示,上表1),使用SYBR® Green Real Time PCR Master Mix Kit试剂盒进行qPCR以检测GH基因是否在草鱼细胞内进行过表达。
结果如图3所示,在三种组织中均呈现类似的结果:两个对照组之间GH基因表达水平差异不显著,而三个实验组相比两个对照组有显著增加,表明碳纳米管基因载体递送***可以使GH基因在草鱼细胞内进行过表达。
5、生长数据分析
在浸泡前一天测量每条实验鱼的体重,记录初始生长数据。在喂养40天后,测量每一条实验鱼的体重,记录实验后的生长数据。比较实验前后草鱼各个处理组的生长差异。上述实验的结果如表2所示。
表2 实验前后草鱼各个处理组生长差异
组别 | 初始体重/g | 生长后体重/g | 增重率/% |
空白对照组 | 2.20 | 2.69 | 22.3 |
SWCNTs对照组 | 2.26 | 2.65 | 17.3 |
实验组1 | 2.23 | 3.47 | 55.6 |
实验组2 | 2.28 | 3.56 | 56.1 |
实验组3 | 2.36 | 3.52 | 49.2 |
由上述结果结合图4可知,实验前每组鱼的体重平均值在2.20~2.36 g之间,各组之间没有显著性差异,在连接有重组质粒的碳纳米管的水体浸泡8小时之后转入正常水体饲养40天,生长后体重相比生长前显著增加,实验后每组鱼的体重平均值在2.69~3.56 g之间,三个实验组之间差异不显著,两个对照组之间差异不显著,而三个实验组相比两个对照组有显著差异,证明经过实验处理可以加快草鱼生长。
综上所述,采用本发明方法能促进草鱼的生长,且浸泡的方式对鱼类的伤害较小,可以提高鱼苗存活率,更容易实现大规模化,经济适用。为促进鱼类生长提供新的思路方法。
本发明可用其他的不违背本发明的精神或主要特征的具体形式来概述。本发明的上述实施例都只能认为是对本发明的说明而不是限制。因此凡是依据本发明的实质技术对以上实施例所作的任何细微修改、等同变化与修饰,均属于本发明技术方案的范围内。
Claims (8)
1.一种采用碳纳米管基因载体递送***促进草鱼生长的方法,其特征在于包括以下步骤:
(S1)构建草鱼生长激素基因重组质粒;
(S2)对碳纳米管进行功能化修饰;
(S3)用超声波将功能化修饰碳纳米管分散于水中,将功能化修饰碳纳米管与水体中草鱼生长激素基因重组质粒结合,得碳纳米管基因载体递送***;
(S4)将碳纳米管基因载体递送***溶于水中,将草鱼在碳纳米管基因载体递送***水溶液中浸泡,通过浸泡的方式将碳纳米管基因载体递送***进入草鱼细胞,使草鱼生长激素基因在草鱼细胞内进行过表达,从而促进草鱼的生长。
2.根据权利要求1所述采用碳纳米管基因载体递送***促进草鱼生长的方法,其特征在于步骤(S1)中构建草鱼生长激素基因重组质粒包括:取草鱼垂体组织样品进行总RNA的提取,反转录得到cDNA,设计引物克隆得到目的基因草鱼GH基因,将pcDNA4.0质粒双酶切后与目的基因草鱼GH基因进行连接得到目的基因重组质粒pcDNA4.0-草鱼GH基因,其中所述引物包括上游引物GH-F和下游引物GH-R,所述上游引物GH-F的序列如SEQ ID NO:2所示,下游引物GH-R的序列如SEQ ID NO:3所示。
3.根据权利要求1所述采用碳纳米管基因载体递送***促进草鱼生长的方法,其特征在于步骤(S2)中对碳纳米管进行功能化修饰包括:用浓硫酸与浓硝酸的混合酸将碳纳米管进行氧化,使其增加官能团羧基,随后与大分子物质聚乙烯亚胺PEI进行氨化反应结合,所得产物经洗涤后冻干得到功能化修饰的碳纳米管。
4.根据权利要求3所述采用碳纳米管基因载体递送***促进草鱼生长的方法,其特征在于:所述浓硫酸与浓硝酸的体积比为2:1~4:1,氧化时间为45~50 h,所述聚乙烯亚胺PEI的分子量为900~1800 Da,氨化时间为70~75 h,所述碳纳米管为单壁碳纳米管,所述单壁碳纳米管的规格为:OD值:1~2 nm;长度:1~3 μm;纯度:>90%。
5.根据权利要求1所述采用碳纳米管基因载体递送***促进草鱼生长的方法,其特征在于:步骤(S3)中用超声波将功能化修饰碳纳米管分散于水中的时间为30~60min。
6.根据权利要求1所述采用碳纳米管基因载体递送***促进草鱼生长的方法,其特征在于:步骤(S3)中所述功能化修饰碳纳米管与草鱼生长激素基因重组质粒的质量份配比为2~8:1。
7.根据权利要求1所述采用碳纳米管基因载体递送***促进草鱼生长的方法,其特征在于:步骤(S4)中所述碳纳米管基因载体递送***水溶液的浓度为1~10 mg/L。
8.根据权利要求1所述采用碳纳米管基因载体递送***促进草鱼生长的方法,其特征在于:步骤(S4)中将草鱼在碳纳米管基因载体递送***水溶液中浸泡6~12 h。
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