CN1354253A - 淡色库蚊胰蛋白酶基因 - Google Patents

淡色库蚊胰蛋白酶基因 Download PDF

Info

Publication number
CN1354253A
CN1354253A CN 01138030 CN01138030A CN1354253A CN 1354253 A CN1354253 A CN 1354253A CN 01138030 CN01138030 CN 01138030 CN 01138030 A CN01138030 A CN 01138030A CN 1354253 A CN1354253 A CN 1354253A
Authority
CN
China
Prior art keywords
gene
culex
dilutus
trypsase
trypsase gene
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN 01138030
Other languages
English (en)
Inventor
马磊
朱昌亮
沈波
田海生
李秀兰
胡莺
杨明夏
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nanjing University
Nanjing Medical University
Original Assignee
Nanjing Medical University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Nanjing Medical University filed Critical Nanjing Medical University
Priority to CN 01138030 priority Critical patent/CN1354253A/zh
Publication of CN1354253A publication Critical patent/CN1354253A/zh
Pending legal-status Critical Current

Links

Landscapes

  • Enzymes And Modification Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

本发明属于基因工程领域,所述淡色库蚊胰蛋白酶基因(Culex pipiens pallens trypsin)基因全长为909bp,其开放阅读框架为783bp,编码261个氨基酸。该基因在GENBANK中的编号为AY034060。本发明应用抑制性差减杂交(suppression substraction hybridization,SSH)结合基因芯片技术分离,并采用快速末端扩增法(rapid amplification cDNAends,RACE)筛选淡色库蚊溴氰菊酯抗性品系cDNA表达文库获得。利用该基因制备融合蛋白及特异性抗体,用于制备与抗药性治理有关的药物及试剂盒。

Description

淡色库蚊胰蛋白酶基因
一.技术领域
本发明属于基因工程领域。
二.背景技术及发明内容
本发明所述淡色库蚊胰蛋白酶基因是发明人在南京医科大学病原生物学系昆虫学实验室应用抑制性差减杂交(suppression substractionhybridization，SSH)结合基因芯片技术分离，并采用快速末端扩增法(rapid amplification cDNA ends，RACE)筛选淡色库蚊溴氰菊酯抗性品系cDNA表达文库获得。序列经拼接获得淡色库蚊胰蛋白酶基因cDNA全长，为909bp；其开放阅读框架为783bp，编码261个氨基酸。查阅GENBANK，其为新基因。该基因在抗溴氰菊酯淡色库蚊中高表达，因此推测淡色库蚊胰蛋白酶基因可能与淡色库蚊溴氰菊酯抗药性相关。
由于淡色库蚊胰蛋白酶基因在GENBANK数据库中为新基因，本发明人对该基因的结构和功能研究成果均为开创性的。美国国家生物技术情报中心(National Center for Biotechnology Information，NCBI)已正式接收该基因为GENBANK的新成员，接收号为AY034060。
淡色库蚊胰蛋白酶基因核苷酸序列：909bp1  UCGAGGGCCGAAUUCCGUUGCUGUCGACUUCCUCUCGUCCGGUAAACCGAAUCCAACGCA 6061 AUGGCCAAGUUAUUAGUGUUGACCACCUGUGCCCUUCUGGGCUUAACAUCUGGUGCCUCC 120121 CUCAAGUCCACCUUGAUGCCGAGCUUUUCGCGCGCAGGGAAAAUCGUCGGAGGGUUCCAG 180181 AUCGACGUCGUCGACGUCCCGUACCAGGUGUCGCUGCAGCGCAAUAACCGUCACCACUGC 240241 GGCGGAUCGAUUAUCGACGAGAGAUGGGUGCUGACGGCGGCCCACUGUACGGAGAAUACC 300301 GACGCCGGUAUCUACAGUGUGCGCGUCGGUUCGUCGGUACACGCCACCGGAGGACAGCUG 360361 GUCCCGGUGAAGGCCGUCCACAACCAUCCGGACUAUGAUCGCGAGGUCACCGAGUUUGAC 420421 UUUUGCUUGCUGGAGUUGGGCGAGCGUUUGGAGUUUGGCCACGCCGUUCAACCGGUUGAU 480481 CUGGUGCGGGACGAACCGGCUGACGAGAGUCAGUCGCUGGUUUCCGGCUGGGGAGACACG 540541 AGAUCGCUGGAGGAAUCCACCGAUAUCCUGAGGGGUGUUUUAGUUCCGUUAGUGAACCGC 600601 GAGGAAUGUGCCGAAGCUUACCAGAAGCUUGGUAUGCCGGUUACGGAGAGCAUGAUCUGC 660661 GCUGGAUUCGCCAAGGAAGGAGUCAAGGACGCCUGCCAAGGGGACAGCGGUGGUCCCCUG 720721 GUCGUGGACGGUCAACUGGCUGGAGUAGUAUCCUGGGGAAAGGGUUGCGCUGAACCUGGA 780781 UAUCCGGGAAUUUACUCCAAUGUUGUGUACGUCCGCGGUUGGAUCAAAAAGGUGGCCAAG 840841 GUUUAAAAACCCAAUAAAGUCUUAAAAUUUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 900901 AAAAAAAAA 909淡色库蚊胰蛋白酶基因氨基酸序列：261aaMAKLLVLTTCALLGLTSGASLKSTLMPSFSRAGKIVGGFQIDVVDVPYQVSLQRNNRHHCGGSIIDERWVLTAAHCTENTDAGIYSVRVGSSVHATGGQLVPVKAVHNHPDYDREVTEFDFCLLELGERLEFGHAVQPVDLVRDEPADESQSLVSGWGDTRSLEESTDILRGVLVPLVNREECAEAYQKLGMPVTESMICAGFAKEGVKDACQGDSGGPLVVDGQLAGVVSWGKGCAEPGYPGIYSNVVYVRGWIKKVAKV
淡色库蚊胰蛋白酶基因是本发明人发现的全新基因，目前人类对于此基因的认识和了解仅限于本发明人所做的研究工作。由于该基因在抗溴氰菊酯淡色库蚊中高表达，因此该基因很可能与淡色库蚊溴氰菊酯抗药性发生密切相关，其表达量的上调可能引起抗药性。该基因可用于：
①通过制备该基因的融合蛋白，研究该基因与抗药性的关系，为昆虫抗药性治理提供新的思路。
②制备特异性抗体，用于野外蚊虫抗药性的监测。现阶段野外蚊虫抗药性大规模检测尚无行之有效的方法，可用该基因编码蛋白的特异性抗体制成试剂盒，可大大简化操作步骤。
三.本发明的技术方案
1.用抑制性差减杂交分离淡色库蚊溴氰菊酯抗性相关基因
取羽化3天内未吸血的淡色库蚊雌性成蚊，分别以对溴氰菊酯抗性品系、敏感品系为检测组(tester)、对应的为驱逐组(driver)，进行正向、反向SSH，以获取抗性相关高表达基因。分别提取两品系蚊虫的总RNA、mRNA，反转录合成cDNA第一链，置换法合成第二链，然后经RsaI酶切、接头连接、2轮差减杂交和2轮PCR，获得正、反向差减杂交产物。杂交产物纯化后将克隆入质粒表达载体PinPointTMXa-1 T-Vector，转化入大肠杆菌JM109。正、反向SSH分别获得523和286个差减克隆，质粒插入片段大小约300-600bp。
2.基因芯片鉴定克隆在抗性品系和敏感品系中的表达差异用PCR扩增获得的809个差减克隆，PCR产物经浓缩、变性处理，用自动点样仪点制于尼龙膜载体上，制成cDNA芯片。提取抗性品系和敏感品系mRNA，反转录后经同位素33P标记为抗性探针和敏感探针。将两种探针分别与2张相同的芯片杂交，通过洗膜、压片、放射自显影和信号扫描获取量化的杂交信号，cDNA芯片共获得差异表达克隆221个。其中在抗性品系中胰蛋白酶比敏感品系表达高3.2倍。
3.构建淡色库蚊抗性品系cDNA表达文库
以淡色库蚊对溴氰菊酯的抗性品系为样本，提取总RNA和mRNA，反转录合成第一、二链。经过EcoRI接头连接、片段的磷酸化和NotI酶切等步骤，使cDNA片段的3’和5’两端分别为NotI和EcoRI粘端。将这些带有粘性末端的cDNA片段定向克隆入λExCell/NotI/EcoRI/CIP噬菌体载体，蛋白质包装使之成为有感染活性的噬菌体，最后将其转染宿主菌一大肠杆菌NM522。淡色库蚊抗性品系cDNA表达文库库容量为4.72×105个重组子，cDNA片段与载体重组率为100％。文库插入片段大小平均约1.1kb。
4.RACE获得淡色库蚊胰蛋白酶基因
采用快速扩增cDNA末端法筛选已构建的cDNA文库。根据已测定的淡色库蚊胰蛋白酶基因片段的序列设计2个引物，分别与插入片段两侧的文库载体序列进行PCR扩增，获取胰蛋白酶基因的5’和3’RACE序列，然后通过T/A克隆插入Pinpoint质粒载体，经过测序、拼接获取全长序列。淡色库蚊胰蛋白酶基因全长序列(GenBank/NCBI AY034060)，总长度为909bp，开放阅读框为783bp，推导的蛋白质编码261个氨基酸。
尽管从原核生物到真核生物，全球科学家已就多物种的胰蛋白酶基因进行了研究，但这是首次获得的在淡色库蚊抗药性品系体内高表达的胰蛋白酶全长基因，这对进一步研究昆虫抗药性机制及其治理具有重要意义。
1.淡色库蚊胰蛋白酶基因核苷酸序列：909bp1  UCGAGGGCCGAAUUCCGUUGCUGUCGACUUCCUCUCGUCCGGUAAACCGAAUCCAACGCA 6061 AUGGCCAAGUUAUUAGUGUUGACCACCUGUGCCCUUCUGGGCUUAACAUCUGGUGCCUCC 120121 CUCAAGUCCACCUUGAUGCCGAGCUUUUCGCGCGCAGGGAAAAUCGUCGGAGGGUUCCAG 180181 AUCGACGUCGUCGACGUCCCGUACCAGGUGUCGCUGCAGCGCAAUAACCGUCACCACUGC 240241 GGCGGAUCGAUUAUCGACGAGAGAUGGGUGCUGACGGCGGCCCACUGUACGGAGAAUACC 300301 GACGCCGGUAUCUACAGUGUGCGCGUCGGUUCGUCGGUACACGCCACCGGAGGACAGCUG 360361 GUCCCGGUGAAGGCCGUCCACAACCAUCCGGACUAUGAUCGCGAGGUCACCGAGUUUGAC 420421 UUUUGCUUGCUGGAGUUGGGCGAGCGUUUGGAGUUUGGCCACGCCGUUCAACCGGUUGAU 480481 CUGGUGCGGGACGAACCGGCUGACGAGAGUCAGUCGCUGGUUUCCGGCUGGGGAGACACG 540541 AGAUCGCUGGAGGAAUCCACCGAUAUCCUGAGGGGUGUUUUAGUUCCGUUAGUGAACCGC 600601 GAGGAAUGUGCCGAAGCUUACCAGAAGCUUGGUAUGCCGGUUACGGAGAGCAUGAUCUGC 660661 GCUGGAUUCGCCAAGGAAGGAGUCAAGGACGCCUGCCAAGGGGACAGCGGUGGUCCCCUG 720721 GUCGUGGACGGUCAACUGGCUGGAGUAGUAUCCUGGGGAAAGGGUUGCGCUGAACCUGGA 780781 UAUCCGGGAAUUUACUCCAAUGUUGUGUACGUCCGCGGUUGGAUCAAAAAGGUGGCCAAG 840841 GUUUAAAAACCCAAUAAAGUCUUAAAAUUUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 900901 AAAAAAAAAA 909
2.淡色库蚊胰蛋白酶基因氨基酸序列：261aaMAKLLVLTTCALLGLTSGASLKSTLMPSFSRAGKIVGGFQIDVVDVPYQVSLQRNNRHHCGGSIIDERWVLTAAHCTENTDAGIYSVRVGSSVHATGGQLVPVKAVHNHPDYDREVTEFDFCLLELGERLEFGHAVQPVDLVRDEPADESQSLVSGWGDTRSLEESTDILRGVLVPLVNREECAEAYQKLGMPVTESMICAGFAKEGVKDACQGDSGGPLVVDGGLAGVVSWGKGCAEPGYPGIYSNVVYVRGWIKKVAKV

Claims (4)

1.一种淡色库蚊胰蛋白酶基因，其特征是淡色库蚊胰蛋白酶基因氨基酸序列为：MAKLLVLTTCALLGLTSGASLKSTLMPSFSRAGKIVGGFQIDVVDVPYQVSLQRNNRHHCGGSIIDERWVLTAAHCTENTDAGIYSVRVGSSVHATGGQLVPVKAVHNHPDYDREVTEFDFCLLELGERLEFGHAVQPVDLVRDEPADESQSLVSGWGDTRSLEESTDILRGVLVPLVNREECAEAYQKLGMPVTESMICAGFAKEGVKDACQGDSGGPLVVDGQLAGWSWGKGCAEPGYPGIYSNVVYVRGWIKKVAKV
2.一种淡色库蚊胰蛋白酶基因，其特征是应用抑制性差减杂交，结合基因芯片技术分离，并采用快速末端扩增法筛选淡色库蚊溴氰菊酯抗性品系cDNA表达文库获得。
3.一种淡色库蚊胰蛋白酶基因制备的融合蛋白，其特征是融合蛋白可用作制备治理昆虫抗药性的有关药物。
4.一种淡色库蚊胰蛋白酶基因制备的特异性抗体，其特征是可用该抗体制成试剂盒，用于野外蚊虫抗药性的监测与抗性机理的研究。
CN 01138030 2001-12-24 2001-12-24 淡色库蚊胰蛋白酶基因 Pending CN1354253A (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN 01138030 CN1354253A (zh) 2001-12-24 2001-12-24 淡色库蚊胰蛋白酶基因

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN 01138030 CN1354253A (zh) 2001-12-24 2001-12-24 淡色库蚊胰蛋白酶基因

Publications (1)

Publication Number Publication Date
CN1354253A true CN1354253A (zh) 2002-06-19

Family

ID=4674322

Family Applications (1)

Application Number Title Priority Date Filing Date
CN 01138030 Pending CN1354253A (zh) 2001-12-24 2001-12-24 淡色库蚊胰蛋白酶基因

Country Status (1)

Country Link
CN (1) CN1354253A (zh)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100403897C (zh) * 2006-06-07 2008-07-23 南京医科大学 胰蛋白酶作为有机杀虫剂降解剂的应用
CN101010425B (zh) * 2004-08-13 2010-05-26 霍夫曼-拉罗奇有限公司 多肽的c-末端修饰
CN102154467A (zh) * 2011-01-12 2011-08-17 南京医科大学 蛋白酶体β亚单位6作为蚊对溴氰菊酯抗性检测的靶标的应用
CN103114086A (zh) * 2013-02-18 2013-05-22 重庆市畜牧科学院 一种快速构建差异表达基因文库的方法

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101010425B (zh) * 2004-08-13 2010-05-26 霍夫曼-拉罗奇有限公司 多肽的c-末端修饰
CN100403897C (zh) * 2006-06-07 2008-07-23 南京医科大学 胰蛋白酶作为有机杀虫剂降解剂的应用
CN102154467A (zh) * 2011-01-12 2011-08-17 南京医科大学 蛋白酶体β亚单位6作为蚊对溴氰菊酯抗性检测的靶标的应用
CN102154467B (zh) * 2011-01-12 2012-08-01 南京医科大学 蛋白酶体β亚单位6作为蚊对溴氰菊酯抗性检测的靶标的应用
CN103114086A (zh) * 2013-02-18 2013-05-22 重庆市畜牧科学院 一种快速构建差异表达基因文库的方法
CN103114086B (zh) * 2013-02-18 2014-09-10 重庆市畜牧科学院 一种快速构建差异表达基因文库的方法

Similar Documents

Publication Publication Date Title
Saboori et al. Polymorphism in the Mr 32,000 Rh protein purified from Rh (D)-positive and-negative erythrocytes.
CN110684121B (zh) 一种靶向her2联合表达pd1-mica融合蛋白的嵌合抗原受体及其表达载体和应用
JPS60115528A (ja) ヒトインタ―ロイキン―２蛋白質を含有する抗腫瘍用または免疫機能低下疾患治療用組成物
EP3835425A1 (en) Recombinant vector for producing antigen for diagnosis of african swine fever and use thereof
CN110023333B (zh) 高亲和力的可溶性pd-1分子
Nienhuis et al. [58] Preparation of globin messenger RNA
CN1354253A (zh) 淡色库蚊胰蛋白酶基因
Huisman et al. Hemoglobin Grady: the first example of a variant with elongated chains due to an insertion of residues
Yu et al. Multi-Omic Identification of Venom Proteins Collected from Artificial Hosts of a Parasitoid Wasp
JPH02502877A (ja) ヒト顆粒球―マクロファージコロニー刺激因子およびそのミューティン
CN109337964A (zh) 一种abo血型突变检测位点
CN1385531A (zh) 淡色库蚊糜蛋白酶基因
JP3537487B2 (ja) ムラサキイガイ接着蛋白質遺伝子
CN110845594A (zh) 可增强长牡蛎免疫应答的重组血清淀粉样蛋白a及制备方法
Nakamura et al. Cloning and characterization of partial cDNAs for woodchuck cytokines and CD3ϵ with applications for the detection of RNA expression in tissues by RT‐PCR assay
WO2001085928A1 (fr) Procede de preparation de vecteur de virus
CN101481692A (zh) 无血清制备重组人类凝血因子ix
Liguori et al. Austwickia chelonae in a wild gopher tortoise (gopherus polyphemus) and evidence of positive selection on the diphtheria-like toxin gene
CN113564169B (zh) 一种日本鳗鲡抗菌肽Cathelicidin2基因启动子及其应用
Shivanand et al. Recombinant DNA technology: Applications in the field of biotechnology and crime sciences
Harder et al. Comprehensive profiling of the human fecal proteome from IBD patients with DIA‐MS enables evaluation of disease‐relevant proteins
CN111116738B (zh) 大菱鲆丝氨酸蛋白酶抑制剂h1的重组蛋白及其制备和应用
Zuo et al. mRNA differential display of colonic mucosa cells in ulcerative colitis
CN111748541B (zh) 一种产mdp的酶制剂在维持肠道稳态中的用途
CN108977434A (zh) 鼻腔脱落细胞rna的提取方法

Legal Events

Date Code Title Description
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
C06 Publication
PB01 Publication
C02 Deemed withdrawal of patent application after publication (patent law 2001)
WD01 Invention patent application deemed withdrawn after publication