CN109576291A - 用于克隆鸡acp5基因cds区序列的引物 - Google Patents

用于克隆鸡acp5基因cds区序列的引物 Download PDF

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CN109576291A
CN109576291A CN201811541386.1A CN201811541386A CN109576291A CN 109576291 A CN109576291 A CN 109576291A CN 201811541386 A CN201811541386 A CN 201811541386A CN 109576291 A CN109576291 A CN 109576291A
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chicken
acp5
primer
gene
region sequence
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施晓丽
文光林
赵忠海
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Guizhou University
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Guizhou University
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
    • C12Y301/03002—Acid phosphatase (3.1.3.2)

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Abstract

本发明公开了一种用于克隆鸡ACP5基因CDS区序列的引物。本发明设计了一组特异性引物，结合该引物运用RT‑PCR技术对鸡ACP5基因进行PCR扩增，PCR扩增目的片段为鸡的ACP5基因的完整CDS区序列，所获得获得的ACP5基因的完整CDS区序列的准确性高、结果可靠，为构建鸡的ACP5基因的原核、真核表达载体及相关动物模型等研究提供基础材料。本发明通过普通克隆手段获得高GC含量序列克隆，所设引物添加酶切位点，可直接用于其他表达实验，同时对笼养蛋鸡骨代谢治疗、改善具有重要实验室意义。

Description

用于克隆鸡ACP5基因CDS区序列的引物
技术领域
本发明涉及生物技术、分子遗传学领域，尤其是用于克隆鸡ACP5基因CDS区序列的引物。
背景技术
抗酒石酸酸性磷酸酶(Tartrate resistant acid phosphatase,ACP5/TRAP/TRACP)为近年来发现的骨吸收和破骨细胞活性的良好标志物，测定血清中ACP5含量，有助于了解生理条件和各种病理条件下的骨代谢状况。研究表明，ACP5主要存在于巨噬细胞、破骨细胞、Gaucher细胞、红细胞、血小板、脾脏毛状细胞以及单核吞噬细胞中,尤其在肺泡巨噬细胞和破骨细胞中含量尤为丰富。
Hayman等(1996)研究显示，但敲除小鼠ACP5基因后，小鼠会表现出轻度的骨钙化，骨折发生频率上升，且破骨细胞活性也显著降低。Hailling等(2017)和Naghsh等(2016)近两年研究显示，ACP5基因的过表达使小鼠患上轻度骨质疏松症，证实ACP5的含量过高可能会使成骨细胞和骨骼合成的活性上升。洪懿燚(2016)研究表明，老年骨质疏松症患者ACP5的含量较低，说明ACP5基因的表达对骨代谢存在影响。
根据以上研究结果可以推测，血清中ACP5的含量将是检验骨代谢疾病预后的重要指标之一，特别是对研究改善和治疗骨代谢疾病的相关模式生物实验的开展能提供极大的参考价值。同样对于畜牧生产中的畜禽来说，也是很有价值的。目前，关于ACP5基因在笼养蛋鸡骨代谢疾病方面的研究鲜见，且该基因的GC含量相对过高，对于PCR扩增的难度也相应增加。
发明内容
本发明所要解决的技术问题是提供一种用于克隆鸡ACP5基因CDS区序列的引物，用它来克隆鸡ACP5基因CDS区序列，具有准确性高、结果可靠的优点，为构建鸡ACP5基因的原核、真核表达载体及相关动物模型等研究提供基础材料。
本发明是这样实现的：用于克隆鸡ACP5基因CDS区序列的引物，该引物包括引物F和引物R，引物F的序列为：5'-CAGCTGATGCTGCTGCTGCTGCT-3'，R:5'-GTCGACTCAGAGGTCACGGGGC-3'；上述引物序列的下划线，表示其酶切位点。
与现有技术相比，本发明设计了一组特异性引物，结合该引物运用RT-PCR技术对鸡ACP5基因进行PCR扩增，PCR扩增目的片段为鸡的ACP5基因的完整CDS区序列，所获得获得的ACP5基因的完整CDS区序列的准确性高、结果可靠，为构建鸡的ACP5基因的原核、真核表达载体及相关动物模型等研究提供基础材料。本发明通过普通克隆手段获得高GC含量序列克隆，所设引物添加酶切位点，可直接用于其他表达实验，同时对笼养蛋鸡骨代谢治疗、改善具有重要实验室意义。
附图说明
图1鸡ACP5基因酶切检测图；
1为质粒酶切，2为质粒DNA，M为DNA Marker 5000；
图2为鸡ACP5基因测序峰图。
具体实施方式
本发明的实施例：克隆鸡ACP5基因编码区的方法
1、鸡总RNA提取及cDNA的合成
采取鸡肉样组织(或骨组织)液氮速冻，-80℃超低温冰箱保存，用于总RNA抽提，参照TRizol Reagent试剂(life/Invitrogen,USA)说明书进行，将所得总RNA逆转录为cDNA的第一条链，cDNA合成根据Thermo Scientific RevertAid First Strand cDNA Synthesis(K1622)逆转录试剂盒合成，
2、鸡ACP5基因CDS区序列引物设计
欲扩增鸡ACP5基因CDS区序列，根据GenBank发布的原鸡ACP5基因(XM_015302697.1)mRNA序列运用NCBI中Primer5.0引物设计软件进行引物设计，同时引入PvuII酶切位点(加粗及下划线为酶切位点碱基)，引物由上海英潍捷基生物有限公司合成，引物如下：
F:5'-CAGCTGATGCTGCTGCTGCTGCT-3'，R:5'-GTCGACTCAGAGGTCACGGGGC-3'
3、鸡ACP5基因CDS区RT-PCR扩增反应：
反应条件：95℃预变性5min；94℃变性40s，退火35s，72℃延伸60s，32循环；72℃终延伸10min，4℃保存。
反应体系：采用30μL反应体系，其中：上游引物1μL，下游引物1μL，10×TranStartTaq Buffer(北京天根)4μL，2.5mM dNTPs 6μL，TranStart Taq DNA Polymerase 1μL，cDNA模板2μL，加ddH2O补足至30μL。
4、重组质粒的构建及测序
鸡ACP5基因CDS区RT-PCR产物经1.2％琼脂糖凝胶电泳后，采用AXYGEN柱式DNA胶回收试剂盒进行RT-PCR产物切胶回收纯化，纯化产物按照
pUC57载体试剂盒说明书进行操作，采用蓝白斑法筛选克隆产物，最终提取重组质粒送往上海英潍捷基生物有限公司测序。
5、结果验证
将克隆结果进行酶切验证(图1)，同时提取重组质粒送往测序公司验证。结果显示，测序峰图正常(图2)，扩增目的片段长度为966bp(附件1)，其中包含起始密码子ATG和终止密码子TAG，所得片段长度与其他物种ACP5基因CDS区片段同源性校稿，与GenBank发布的原鸡ACP5基因(XM_015302697.1)mRNA序列比对，发现3个SNPs，说明鸡的ACP5基因CDS区序列克隆成功，该结果可用于后续其他实验。
虽然本发明已将实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。
SEQUENCE LISTING
序列表
<110> 贵州大学
<120> 用于克隆鸡ACP5基因CDS区序列的引物
<130> nm:
<160> 2
<170> PatentIn version
<210> 1
<211> 23
<212> DNA
<213> 鸡(Gallus gallus)
<220>
<223>根据GenBank发布的原鸡ACP5基因（XM_015302697.1）为依据，利用Primer6.0设计PCR引物,以用于PCR扩增。
<400> 1
cagct gatgc tgctg ctgct gct 23
<210> 2
<211> 22
<212> DNA
<213> 鸡(Gallus gallus)
<220>
<223>根据GenBank发布的原鸡ACP5基因（XM_015302697.1）为依据，利用Primer6.0设计PCR引物,以用于PCR扩增。
<400> 2
gtcga ctcag aggtc acggg gc 22

Claims (1)

1.一种用于克隆鸡ACP5基因CDS区序列的引物，其特征在于：该引物包括引物F和引物R，引物F的序列为：5'-CAGCTGATGCTGCTGCTGCTGCT-3'，R:5'-GTCGACTCAGAGGTCACGGGGC-3'；上述引物序列的下划线，表示其酶切位点。
CN201811541386.1A 2018-12-17 2018-12-17 用于克隆鸡acp5基因cds区序列的引物 Pending CN109576291A (zh)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030175749A1 (en) * 2001-12-08 2003-09-18 Jong-Yoon Chun Annealing control primer and its uses
WO2003093509A1 (en) * 2002-05-01 2003-11-13 Seegene, Inc. Methods and compositions for improving specificity of pcr amplication
CN105002185A (zh) * 2015-08-14 2015-10-28 贵州大学 一种克隆白洗猪pid1基因cds区序列的方法
CN105002164A (zh) * 2015-08-14 2015-10-28 贵州大学 一种克隆猪lyrm1基因cds区序列的方法
WO2017214828A1 (zh) * 2016-06-14 2017-12-21 石庆学 特异促进 Prkcz 基因高表达的慢病毒表达载体及其应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030175749A1 (en) * 2001-12-08 2003-09-18 Jong-Yoon Chun Annealing control primer and its uses
WO2003093509A1 (en) * 2002-05-01 2003-11-13 Seegene, Inc. Methods and compositions for improving specificity of pcr amplication
CN105002185A (zh) * 2015-08-14 2015-10-28 贵州大学 一种克隆白洗猪pid1基因cds区序列的方法
CN105002164A (zh) * 2015-08-14 2015-10-28 贵州大学 一种克隆猪lyrm1基因cds区序列的方法
WO2017214828A1 (zh) * 2016-06-14 2017-12-21 石庆学 特异促进 Prkcz 基因高表达的慢病毒表达载体及其应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
佚名: "XM_015302697.2 PREDICTED: Gallus gallus acid phosphatase 5, tartrate resistant (ACP5), mRNA" *

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