中华蜜蜂幼虫膜蛋白酵母双杂交文库的构建
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摘要
【目的】本研究旨在利用位点特异性重组技术(FullCoV)将中华蜜蜂Apis cerana cerana幼虫膜蛋白cDNA连接到pPR3-N载体上,构建中华蜜蜂幼虫膜蛋白酵母双杂交cDNA文库。【方法】提取2-3日龄中华蜜蜂工蜂幼虫总RNA;分离mRNA后,在反转录酶的作用下合成幼虫膜蛋白cDNA第1链,并合成双链cDNA。在双链cDNA的5′端加上带有重组序列的接头后,通过FullCoV技术与载体pPR3-N进行连接,然后将连接产物电转化到DH10B感受态细胞,构建中华蜜蜂幼虫膜蛋白酵母双杂交cDNA文库,并对该文库插入片段大小和文库滴度进行检测。【结果】通过FullCoV技术成功构建了中华蜜蜂幼虫膜蛋白酵母双杂交cDNA文库,经检测,中华蜜蜂幼虫膜蛋白酵母cDNA文库的总库容量为1.5×10~7 cfu,文库滴度为3×10~6 cfu/mL,重组率达到100%。【结论】本研究利用FullCoV技术成功构建了中华蜜蜂幼虫膜蛋白酵母cDNA文库,为进一步探究感染中华蜜蜂的病原微生物与宿主蛋白互作研究奠定了基础。
【Aim】 This study aims to construct the yeast two-hybrid cDNA library for membrane proteins of Apis cerana cerana larvae by cloning the larval membrane protein cDNA into pPR3-N using FullCoV technique. 【Methods】 The total RNA was extracted from the 2-3 day-old worker larvae of A. cerana cerana. After mRNA was isolated, the first strand cDNA of membrane protein was synthesized by reverse transcriptase, and then the double-stranded cDNA was synthesized. After being added to the 5′ end of the double-stranded cDNA, the linker with the recombinant sequence was ligated with the vector pPR3-N by FullCoV technology. The ligation product was electrotransformed into DH10 B competent cells to construct the larval membrane protein cDNA library, and the titer of the library and the size of the inserted cDNA fragment were examined and verified. 【Results】 A yeast two-hybrid cDNA library for membrane proteins of A. cerana cerana larvae was successfully constructed by the FullCoV technique. The library had the capacity of 1.5×10~7 cfu, the titer of 3×10~6 cfu/mL, and the recombination rate of 100%. 【Conclusion】 In this study the yeast two-hybrid cDNA library for membrane proteins of A. cerana cerana larvae was successfully constructed using FullCoV technique, which is helpful for the further study on the interaction between pathogen infecting A. cerana cerana and host proteins.
【Aim】 This study aims to construct the yeast two-hybrid cDNA library for membrane proteins of Apis cerana cerana larvae by cloning the larval membrane protein cDNA into pPR3-N using FullCoV technique. 【Methods】 The total RNA was extracted from the 2-3 day-old worker larvae of A. cerana cerana. After mRNA was isolated, the first strand cDNA of membrane protein was synthesized by reverse transcriptase, and then the double-stranded cDNA was synthesized. After being added to the 5′ end of the double-stranded cDNA, the linker with the recombinant sequence was ligated with the vector pPR3-N by FullCoV technology. The ligation product was electrotransformed into DH10 B competent cells to construct the larval membrane protein cDNA library, and the titer of the library and the size of the inserted cDNA fragment were examined and verified. 【Results】 A yeast two-hybrid cDNA library for membrane proteins of A. cerana cerana larvae was successfully constructed by the FullCoV technique. The library had the capacity of 1.5×10~7 cfu, the titer of 3×10~6 cfu/mL, and the recombination rate of 100%. 【Conclusion】 In this study the yeast two-hybrid cDNA library for membrane proteins of A. cerana cerana larvae was successfully constructed using FullCoV technique, which is helpful for the further study on the interaction between pathogen infecting A. cerana cerana and host proteins.
引文
Bashline L,Gu Y,2014.Using the split-ubiquitin yeast two-hybrid system to test protein-protein interactions of transmembrane proteins.Meth.Mol.Biol.,1242:143-158.
Berényi O,Bakonyi T,Derakhshifar I,K?glberger H,Nowotny N,2006.Occurrence of six honeybee viruses in diseased Austrian apiaries.Appl.Environ.Microbiol.,72(4):2414-2420.
Cox C,McKenna JP,Watt AP,Coyle PV,2016.Urea plasma parvum and mycoplasma genitalium are found to be significantly associated with microscopy-confirmed urethritis in a routine genitourinary medicine setting.Int.J.STD AIDS,27(10):861-867.
Danismazoglu M,Nalcacioglu R,Muratoglu H,Demirbag Z,2018.The protein-protein interactions between Amsacta moorei entomopoxvirus (AMEV) protein kinases (PKs) and all viral proteins.Virus Res.,248:31-38.
Du W,Xia J,Zang Y,Liu MJ,Li HB,Yan XM,Zhang JS,Li N,Zhou ZY,Xie XZ,2015.Expression of recombinant myostatin propeptide pPIC9K-Msp plasmid in Pichia pastoris.Genet.Mol.Res.,14(4):18414-18420.
Indriolo E,Goring DR,2016.Yeast two-hybrid interactions between Arabidopsis lyrata S Receptor Kinase and the ARC1 E3 ligase.Plant Signa.Behav.,11(6):e1188233.
Ivanusic D,Heinisch JJ,Eschricht M,Laube U,Denner J,2015.Improved split-ubiquitin screening technique to identify surface membrane protein-protein interactions.BioTechniques,59(2):63-73.
Kittanakom S,Chuk M,Wong V,Snyder J,Edmonds D,Lydakis A,Zhang Z,2009.Analysis of membrane protein complexes using the split-ubiquitin membrane yeast two-hybrid (MYTH) system.Methods Mol.Biol.,548:247-271.
Li J,Gao J,Han L,Zhang Y,Guan W,Zhou L,Yu Y,Han W,2016.Development of a membrane-anchored ligand and receptor yeast two-hybrid system for ligand-receptor interaction identification.Sci.Rep.,6:35631.
Li QP,Wang S,Gou JY,2017.A split ubiquitin system to reveal topology and released peptides of membrane proteins.BMC Biotechnol.,17(1):69.
Liao YJ,Li S,He F,He WR,Dong H,Feng S,Sun Y,Qiu HJ,2013.Construction of cDNA expression library of porcine peripheral blood mononuclear cells and screening of cell proteins interacting with classical swine fever virus E2 protein.Chin.J.Prev.Veter.Med.,35(9):707-710.[廖亚金,李素,贺番,何文瑞,董泓,冯硕,孙元,仇华吉,2013.猪外周血单个核细胞cDNA酵母表达文库的构建及与猪瘟病毒E2蛋白相互作用细胞蛋白的筛选.中国预防兽医学报,35(9):707-710]
Liu X,Zhang Y,Yan X,Han R,2010.Prevention of Chinese sacbrood virus infection in Apis cerana using RNA interference.Curr.Microbiol.,61(5):422-428.
Ma M,2014.New insights of sacbrood virus.Virol.Sin.,29(6):410-413.
Marik A,Naiya H,Das M,Mukherjee G,Basu S,Saha C,Chowdhury R,Bhattacharyya K,Seal A,2016.Split-ubiquitin yeast two-hybrid interaction reveals a novel interaction between a natural resistance associated macrophage protein and a membrane bound thioredoxin in Brassica juncea.Plant Mol.Biol.,92(4-5):519-537.
Martin DH,Manhart LE,Workowski KA,2017.Mycoplasma genitalium from basic science to public health:summary of the results from a national institute of allergy and infectious diseases technical consultation and consensus recommendations for future research priorities.J.Infect.Dis.,216(Suppl_2):S427-S430.
Memi?evi.Mining host-pathogen protein interactions to characterize Burkholderia mallei infectivity mechanisms.PLoS Comp.Biol.,11(3):e1004088.
Petschnigg J,Wong V,Snider J,Stagljar I,2012.Investigation of membrane protein interactions using the split-ubiquitin membrane yeast two-hybrid system.Methods Mol.Biol.,812:225-244.
Snider J,Stagljar I,2016.Membrane yeast two-hybrid (MYTH) mapping of full-length membrane protein interactions.Cold Spring Harb.Protoc.,(1):pdb.top077560.
Tantillo G,Bottaro M,Pinto AD,Vito M,Pietro DP,Valentina T,2015.Virus infections of honeybees Apis mellifera.Ital.J.Food Saf.,4(3):5364.
Wang B,Pelletier J,Massaad MJ,Herscovics A,Shore GC,2004.The yeast split-ubiquitin membrane protein two-hybrid screen identifies BAP31 as a regulator of the turnover of endoplasmic reticulum-associated protein tyrosine phosphatase-like B.Mol.Cell.Biol.,24(7):2767-2778.
Wei D,2017.Construction of Yeast Two-hybrid cDNA Library for Apis cerana Larvae and Screening Its Interaction Protein with VP3 of Sacbrood Virus.MSc Thesis,Jinzhou Medical University,Jinzhou,Liaoning.[魏东,2017.中蜂幼虫酵母双杂交文库构建及其与囊状幼虫病毒VP3互作蛋白筛选.辽宁锦州:锦州医科大学硕士学位论文]
Yan X,Han RC,2008.Diagnostic technologies of common pathogens of honeybees in China.Chin.Bull.Entomol.,45(3):483-488.[颜珣,韩日畴,2008.我国蜜蜂主要病原检测技术.昆虫知识,45(3):483-488]
Yanatori I,Yasui Y,Ouchi K,Kishi F,2015.Chlamydia pneumoniae CPj0783 interaction with Huntingtin-protein14.Int.Microbiol.,18(4):225-233.
Yasuyuki N,Jun I,Akihiko K,2013.Rapid,facile detection of heterodimer partners for target human G-protein-coupled receptors using a modified split-ubiquitin membrane yeast two-hybrid system.PLoS ONE,8(6):e66793.
Yuan WF,Wu BM,Zhang XY,Zhang Q,Sun HC,2009.Preliminary screening of IBDV VP2-binding protein by yeast two-hybrid system.Acta Veter.Zootech.Sin.,40(6):958-962.[袁维峰,吴保明,张鑫宇,张泉,孙怀昌,2009.应用酵母双杂交系统初步筛选IBDV VP2结合蛋白.畜牧兽医学报,40(6):958-962]
Berényi O,Bakonyi T,Derakhshifar I,K?glberger H,Nowotny N,2006.Occurrence of six honeybee viruses in diseased Austrian apiaries.Appl.Environ.Microbiol.,72(4):2414-2420.
Cox C,McKenna JP,Watt AP,Coyle PV,2016.Urea plasma parvum and mycoplasma genitalium are found to be significantly associated with microscopy-confirmed urethritis in a routine genitourinary medicine setting.Int.J.STD AIDS,27(10):861-867.
Danismazoglu M,Nalcacioglu R,Muratoglu H,Demirbag Z,2018.The protein-protein interactions between Amsacta moorei entomopoxvirus (AMEV) protein kinases (PKs) and all viral proteins.Virus Res.,248:31-38.
Du W,Xia J,Zang Y,Liu MJ,Li HB,Yan XM,Zhang JS,Li N,Zhou ZY,Xie XZ,2015.Expression of recombinant myostatin propeptide pPIC9K-Msp plasmid in Pichia pastoris.Genet.Mol.Res.,14(4):18414-18420.
Indriolo E,Goring DR,2016.Yeast two-hybrid interactions between Arabidopsis lyrata S Receptor Kinase and the ARC1 E3 ligase.Plant Signa.Behav.,11(6):e1188233.
Ivanusic D,Heinisch JJ,Eschricht M,Laube U,Denner J,2015.Improved split-ubiquitin screening technique to identify surface membrane protein-protein interactions.BioTechniques,59(2):63-73.
Kittanakom S,Chuk M,Wong V,Snyder J,Edmonds D,Lydakis A,Zhang Z,2009.Analysis of membrane protein complexes using the split-ubiquitin membrane yeast two-hybrid (MYTH) system.Methods Mol.Biol.,548:247-271.
Li J,Gao J,Han L,Zhang Y,Guan W,Zhou L,Yu Y,Han W,2016.Development of a membrane-anchored ligand and receptor yeast two-hybrid system for ligand-receptor interaction identification.Sci.Rep.,6:35631.
Li QP,Wang S,Gou JY,2017.A split ubiquitin system to reveal topology and released peptides of membrane proteins.BMC Biotechnol.,17(1):69.
Liao YJ,Li S,He F,He WR,Dong H,Feng S,Sun Y,Qiu HJ,2013.Construction of cDNA expression library of porcine peripheral blood mononuclear cells and screening of cell proteins interacting with classical swine fever virus E2 protein.Chin.J.Prev.Veter.Med.,35(9):707-710.[廖亚金,李素,贺番,何文瑞,董泓,冯硕,孙元,仇华吉,2013.猪外周血单个核细胞cDNA酵母表达文库的构建及与猪瘟病毒E2蛋白相互作用细胞蛋白的筛选.中国预防兽医学报,35(9):707-710]
Liu X,Zhang Y,Yan X,Han R,2010.Prevention of Chinese sacbrood virus infection in Apis cerana using RNA interference.Curr.Microbiol.,61(5):422-428.
Ma M,2014.New insights of sacbrood virus.Virol.Sin.,29(6):410-413.
Marik A,Naiya H,Das M,Mukherjee G,Basu S,Saha C,Chowdhury R,Bhattacharyya K,Seal A,2016.Split-ubiquitin yeast two-hybrid interaction reveals a novel interaction between a natural resistance associated macrophage protein and a membrane bound thioredoxin in Brassica juncea.Plant Mol.Biol.,92(4-5):519-537.
Martin DH,Manhart LE,Workowski KA,2017.Mycoplasma genitalium from basic science to public health:summary of the results from a national institute of allergy and infectious diseases technical consultation and consensus recommendations for future research priorities.J.Infect.Dis.,216(Suppl_2):S427-S430.
Memi?evi.Mining host-pathogen protein interactions to characterize Burkholderia mallei infectivity mechanisms.PLoS Comp.Biol.,11(3):e1004088.
Petschnigg J,Wong V,Snider J,Stagljar I,2012.Investigation of membrane protein interactions using the split-ubiquitin membrane yeast two-hybrid system.Methods Mol.Biol.,812:225-244.
Snider J,Stagljar I,2016.Membrane yeast two-hybrid (MYTH) mapping of full-length membrane protein interactions.Cold Spring Harb.Protoc.,(1):pdb.top077560.
Tantillo G,Bottaro M,Pinto AD,Vito M,Pietro DP,Valentina T,2015.Virus infections of honeybees Apis mellifera.Ital.J.Food Saf.,4(3):5364.
Wang B,Pelletier J,Massaad MJ,Herscovics A,Shore GC,2004.The yeast split-ubiquitin membrane protein two-hybrid screen identifies BAP31 as a regulator of the turnover of endoplasmic reticulum-associated protein tyrosine phosphatase-like B.Mol.Cell.Biol.,24(7):2767-2778.
Wei D,2017.Construction of Yeast Two-hybrid cDNA Library for Apis cerana Larvae and Screening Its Interaction Protein with VP3 of Sacbrood Virus.MSc Thesis,Jinzhou Medical University,Jinzhou,Liaoning.[魏东,2017.中蜂幼虫酵母双杂交文库构建及其与囊状幼虫病毒VP3互作蛋白筛选.辽宁锦州:锦州医科大学硕士学位论文]
Yan X,Han RC,2008.Diagnostic technologies of common pathogens of honeybees in China.Chin.Bull.Entomol.,45(3):483-488.[颜珣,韩日畴,2008.我国蜜蜂主要病原检测技术.昆虫知识,45(3):483-488]
Yanatori I,Yasui Y,Ouchi K,Kishi F,2015.Chlamydia pneumoniae CPj0783 interaction with Huntingtin-protein14.Int.Microbiol.,18(4):225-233.
Yasuyuki N,Jun I,Akihiko K,2013.Rapid,facile detection of heterodimer partners for target human G-protein-coupled receptors using a modified split-ubiquitin membrane yeast two-hybrid system.PLoS ONE,8(6):e66793.
Yuan WF,Wu BM,Zhang XY,Zhang Q,Sun HC,2009.Preliminary screening of IBDV VP2-binding protein by yeast two-hybrid system.Acta Veter.Zootech.Sin.,40(6):958-962.[袁维峰,吴保明,张鑫宇,张泉,孙怀昌,2009.应用酵母双杂交系统初步筛选IBDV VP2结合蛋白.畜牧兽医学报,40(6):958-962]