二斑叶螨几丁质酶基因的原核表达及多克隆抗体制备
以制备的二斑叶螨c DNA为模板,克隆二斑叶螨几丁质酶Se Chi基因功能结构域催化区片段,大小为786 bp。将该序列片段克隆到表达载体p ET-32a（＋）中,获得多克隆原核表达载体p ET-32a-Tu Chi。重组质粒经酶切测序鉴定后转化大肠杆菌BL21（DE3）,经0.6 mmol·L-1IPTG诱导4 h后高效表达约45 ku可溶性重组蛋白;经His亲和层析洗脱和浓缩获得高纯度的重组蛋白免疫新西兰大白兔制备Tu Chi多克隆抗体。制备的多克隆抗体经Western Blot证实能特异性地识别几丁质酶而不与非特异性蛋白结合。ELISA分析表明制备的抗体效价达1？？80 000,效价较高...
Saved in:
| Published in | 东北农业大学学报 Vol. 46; no. 1; pp. 62 - 67 |
|---|---|
| Main Author | |
| Format | Journal Article |
| Language | Chinese |
| Published |
遵义师范学院生命科学学院/赤水河流域动物资源保护与应用研究重点实验室,贵州 遵义,563002%遵义医学院生化与分子生物学教研室,贵州 遵义,563009%贵州省烟草科学研究院,贵阳,550083
2015
|
| Subjects | |
| Online Access | Get full text |
| ISSN | 1005-9369 |
Cover
| Abstract | 以制备的二斑叶螨c DNA为模板,克隆二斑叶螨几丁质酶Se Chi基因功能结构域催化区片段,大小为786 bp。将该序列片段克隆到表达载体p ET-32a（＋）中,获得多克隆原核表达载体p ET-32a-Tu Chi。重组质粒经酶切测序鉴定后转化大肠杆菌BL21（DE3）,经0.6 mmol·L-1IPTG诱导4 h后高效表达约45 ku可溶性重组蛋白;经His亲和层析洗脱和浓缩获得高纯度的重组蛋白免疫新西兰大白兔制备Tu Chi多克隆抗体。制备的多克隆抗体经Western Blot证实能特异性地识别几丁质酶而不与非特异性蛋白结合。ELISA分析表明制备的抗体效价达1？？80 000,效价较高,为进一步研究二斑叶螨几丁质酶相关功能奠定基础。 |
|---|---|
| AbstractList | 以制备的二斑叶螨c DNA为模板,克隆二斑叶螨几丁质酶Se Chi基因功能结构域催化区片段,大小为786 bp。将该序列片段克隆到表达载体p ET-32a（＋）中,获得多克隆原核表达载体p ET-32a-Tu Chi。重组质粒经酶切测序鉴定后转化大肠杆菌BL21（DE3）,经0.6 mmol·L-1IPTG诱导4 h后高效表达约45 ku可溶性重组蛋白;经His亲和层析洗脱和浓缩获得高纯度的重组蛋白免疫新西兰大白兔制备Tu Chi多克隆抗体。制备的多克隆抗体经Western Blot证实能特异性地识别几丁质酶而不与非特异性蛋白结合。ELISA分析表明制备的抗体效价达1？？80 000,效价较高,为进一步研究二斑叶螨几丁质酶相关功能奠定基础。 Q786; 以制备的二斑叶螨cDNA为模板，克隆二斑叶螨几丁质酶SeChi基因功能结构域催化区片段，大小为786 bp。将该序列片段克隆到表达载体pET-32a (+)中，获得多克隆原核表达载体pET-32a-TuChi。重组质粒经酶切测序鉴定后转化大肠杆菌BL21(DE3)，经0.6 mmol·L-1 IPTG诱导4 h后高效表达约45 ku可溶性重组蛋白；经His亲和层析洗脱和浓缩获得高纯度的重组蛋白免疫新西兰大白兔制备TuChi多克隆抗体。制备的多克隆抗体经Western Blot证实能特异性地识别几丁质酶而不与非特异性蛋白结合。ELISA分析表明制备的抗体效价达1??80000，效价较高，为进一步研究二斑叶螨几丁质酶相关功能奠定基础。 |
| Abstract_FL | The sequence of part chitinase gene of Tetranychus urticae was amplified by PCR from cDNA. The sequence length of this domain was 786 bp. Then, the gene was cloned into pET-32a (+) prokaryotic expressive vector, and the constructed recombinant plasmids pET-32a-TuChi was transformed into the host bacteria E. coli BL21 (DE3). About 45 ku fusion protein was abundantly expressed at 4 h after the recombinant vector was induced with 0.6 mmol·L-1 IPTG. The fusion protein was purified on a Ni+affinity column. The purified chitinase protein was used to immunize New Zealand rabbits for preparing polyclonal antibody with specificity as defined by Western Blot. ELISA analysis showed that the titer of the polyclonal antibody was 1??80 000, polyclonal antibody that was prepared had a high titer and specificity, and the results laid the foundation to further study the function of the chitinase in Tetranychus urticae. |
| Author | 张道伟 陈静 张正玲 曾燕玲 郭玉双 |
| AuthorAffiliation | 遵义师范学院生命科学学院,赤水河流域动物资源保护与应用研究重点实验室,贵州遵义563002 遵义医学院生化与分子生物学教研室,贵州遵义563009 贵州省烟草科学研究院,贵阳550083 |
| AuthorAffiliation_xml | – name: 遵义师范学院生命科学学院/赤水河流域动物资源保护与应用研究重点实验室,贵州 遵义,563002%遵义医学院生化与分子生物学教研室,贵州 遵义,563009%贵州省烟草科学研究院,贵阳,550083 |
| Author_FL | ZHANG Daowei CHEN Jing ZENG Yanling ZHANG Zhengling GUO Yushuang |
| Author_FL_xml | – sequence: 1 fullname: ZHANG Daowei – sequence: 2 fullname: CHEN Jing – sequence: 3 fullname: ZHANG Zhengling – sequence: 4 fullname: ZENG Yanling – sequence: 5 fullname: GUO Yushuang |
| Author_xml | – sequence: 1 fullname: 张道伟 陈静 张正玲 曾燕玲 郭玉双 |
| BookMark | eNotjU1LAkEch-dgkJlfoluHhfk7bzvHEHsBoYt32XVmbaPGUiI9BplhkkRQYJAGHvYDxCJCfhrH1m_RgvE7PJeH57eDMqZhdAZlAWPmSMLlNsq3WqGPCadEAIgsKi3ng9Xbix3GyWdke5Pl7C75jtbd2I7n9mPyO7q3z-PVZJZ8RcliYYd9Ox3Z7tN69LDqvy9_Xu1jbKe9XbQVeBctnf9nDlUOS5XisVM-PTopHpSdGgfsKEEZpUClUCkCDZrowHW5xzhT2vW1hkJNS0kYJUQqIEGBScG54hhcIQXJof1N9tYzgWfq1fPGTdOkh1Xlm45qt_0CBoYhXerubdzaWcPUr8PUvmqGl16zU-WcuC6VAOQPs-dsNg |
| ClassificationCodes | Q786 |
| ContentType | Journal Article |
| Copyright | Copyright © Wanfang Data Co. Ltd. All Rights Reserved. |
| Copyright_xml | – notice: Copyright © Wanfang Data Co. Ltd. All Rights Reserved. |
| DBID | 2RA 92L CQIGP W95 ~WA 2B. 4A8 92I 93N PSX TCJ |
| DatabaseName | 维普_期刊 中文科技期刊数据库-CALIS站点 维普中文期刊数据库 中文科技期刊数据库-农业科学 中文科技期刊数据库- 镜像站点 Wanfang Data Journals - Hong Kong WANFANG Data Centre Wanfang Data Journals 万方数据期刊 - 香港版 China Online Journals (COJ) China Online Journals (COJ) |
| DatabaseTitleList | |
| DeliveryMethod | fulltext_linktorsrc |
| DocumentTitleAlternate | Expression and polyclonal antibody preparation of chitinase gene in Tetranychus urticae Koch |
| DocumentTitle_FL | Expression and polyclonal antibody preparation of chitinase gene in Tetranychus urticae Koch |
| EndPage | 67 |
| ExternalDocumentID | dbnydxxb201501010 663884911 |
| GrantInformation_xml | – fundername: 贵州省科技厅项目; 遵义师范学院博士基金 funderid: (黔科合J字[2013]2307号); (BSJJ201206) |
| GroupedDBID | 2RA 92L ALMA_UNASSIGNED_HOLDINGS CDYEO CQIGP W95 ~WA 2B. 4A8 92I 93N PSX TCJ |
| ID | FETCH-LOGICAL-c610-d745441497d441fe1e3ef886a565de8bee12ce99354339d13f259766d60187973 |
| ISSN | 1005-9369 |
| IngestDate | Thu May 29 04:07:26 EDT 2025 Wed Feb 14 10:33:08 EST 2024 |
| IsPeerReviewed | true |
| IsScholarly | true |
| Issue | 1 |
| Keywords | 几丁质酶 chitinase Tetranychus urticae 原核表达 polyclonal antibody 二斑叶螨 prokaryotic expression 多克隆抗体 |
| Language | Chinese |
| LinkModel | OpenURL |
| MergedId | FETCHMERGED-LOGICAL-c610-d745441497d441fe1e3ef886a565de8bee12ce99354339d13f259766d60187973 |
| Notes | ZHANG Daowei;CHEN Jing;ZHANG Zhengling;ZENG Yanling;GUO Yushuang （1. School of Life Sciences Zunyi Normal College/Key Laboratory of Protection and Utilization of Animal Resource in Chishui River Basin, Zunyi Guizhou 563002, China 2. Department of Biochemistry and Molecular Biology, Zunyi Medical College, Zunyi Guizhou 563009 China; 3. Guizhou Tobacco Research Institute, Guiyang 550083, China） The sequence of part chitinase gene of Tetranychus urticae was amplified by PCR from c DNA. The sequence length of this domain was 786 bp. Then, the gene was cloned into p ET- 32a（＋）prokaryotic expressive vector, and the constructed recombinant plasmids p ET- 32a- Tu Chi was transformed into the host bacteria E. coli BL21（DE3）. About 45 ku fusion protein was abundantly expressed at 4 h after the recombinant vector was induced with 0.6 mmol· L-1IPTG. The fusion protein was purified on a Ni＋affinity column. The purified chitinase protein was used to immunize New Zealand rabbits for preparing polyclonal antibody with specifi |
| PageCount | 6 |
| ParticipantIDs | wanfang_journals_dbnydxxb201501010 chongqing_primary_663884911 |
| PublicationCentury | 2000 |
| PublicationDate | 2015 |
| PublicationDateYYYYMMDD | 2015-01-01 |
| PublicationDate_xml | – year: 2015 text: 2015 |
| PublicationDecade | 2010 |
| PublicationTitle | 东北农业大学学报 |
| PublicationTitleAlternate | Journal of Northeast Agricultural University |
| PublicationTitle_FL | Journal of Northeast Agricultural University |
| PublicationYear | 2015 |
| Publisher | 遵义师范学院生命科学学院/赤水河流域动物资源保护与应用研究重点实验室,贵州 遵义,563002%遵义医学院生化与分子生物学教研室,贵州 遵义,563009%贵州省烟草科学研究院,贵阳,550083 |
| Publisher_xml | – name: 遵义师范学院生命科学学院/赤水河流域动物资源保护与应用研究重点实验室,贵州 遵义,563002%遵义医学院生化与分子生物学教研室,贵州 遵义,563009%贵州省烟草科学研究院,贵阳,550083 |
| SSID | ssib036437117 ssib012081041 ssib006567412 ssib001100945 ssib000272666 ssib002263419 ssib051368045 |
| Score | 2.0041053 |
| Snippet | 以制备的二斑叶螨c DNA为模板,克隆二斑叶螨几丁质酶Se Chi基因功能结构域催化区片段,大小为786 bp。将该序列片段克隆到表达载体p ET-32a（＋）中,获得多克隆原核表达载体p... Q786; 以制备的二斑叶螨cDNA为模板，克隆二斑叶螨几丁质酶SeChi基因功能结构域催化区片段，大小为786 bp。将该序列片段克隆到表达载体pET-32a (+)中，获得多克隆原核表达载... |
| SourceID | wanfang chongqing |
| SourceType | Aggregation Database Publisher |
| StartPage | 62 |
| SubjectTerms | 二斑叶螨 几丁质酶 原核表达 多克隆抗体 |
| Title | 二斑叶螨几丁质酶基因的原核表达及多克隆抗体制备 |
| URI | http://lib.cqvip.com/qk/90164A/201501/663884911.html https://d.wanfangdata.com.cn/periodical/dbnydxxb201501010 |
| Volume | 46 |
| hasFullText | 1 |
| inHoldings | 1 |
| isFullTextHit | |
| isPrint | |
| journalDatabaseRights | – providerCode: PRVCAB databaseName: Nutrition and Food Sciences Database issn: 1005-9369 databaseCode: DYU dateStart: 20110101 customDbUrl: isFulltext: true dateEnd: 99991231 titleUrlDefault: https://www.cabidigitallibrary.org/product/zd omitProxy: true ssIdentifier: ssib012081041 providerName: CAB International |
| link | http://utb.summon.serialssolutions.com/2.0.0/link/0/eLvHCXMwnR1Na9RANCw9eRFFxVqVIs5piWySmcnMMclmKYKetlBPSz5bPKR-bKH2JlgrVSwiKKxgV-hhf4AspWB_zWbdHvwPvjfJblMRUSFkh_c5895k580kb0bTbovYYjIOuB5KEegwBDA9NGiiR5Ft0SgN4zTABOd79_nSMr27wlZqtR-Vr5Y2uuGdaOu3eSX_41WAgV8xS_YfPDsTCgAog3_hDh6G-1_5mPiUuA4RHvE5kXAZxGdEtIjLiS-I9IkjFMQmTkMRC4gcEeVShZJEMEXMiGyhKCy4itgmEiRTxe4jFlQAHCQAu2ModpDj41UoFYrdociIELhcVIFyOLIDgbRVNZpEWopGlNqBqxwRH1aaBk3wFJmnGBnKQUiBmqpzFMppEodXCkqdw6YdCjGup1om0QaonyJEtuqqkhIrgwWom6xXyLmSaKFFwBCuWVfGdlW7bTStZFUcyLGQowAJOTWOV11fKXJLy8EAd2nFAw-ro0W5YFp9Koq__nJQKYKI4oiRX3byhuBOCCpVJjrEhbhU0HywXH0HbJ6ZTBr42WclmDX5mc33IBK36WlwapgQ3TVOg1sLX8lWsoiZYXHRUKd2zxqGG4qsrWerjyFMUllrWRpkq5UAq31BO1_OjBadoptf1Gpba5c0f3T0ZvzhXb43nHwe5Dv90eHzydfByfYw3z_KP_W_917kb_fH_cPJl8Hk-Djf280Pevn265Pey_Hux9G39_mrYX6wc1lrt_y2t6SXJ3_oEYTzemxTPBqPSjuGnzQxEitJheABzD7iRIRJYphRApE1o5YlY8NKYRJvcx5zPGJS2tYVbS5bz5Kr2mLIAzsUZhIyMB63jcCIg1Qy4IwbzAyDeW1hZoHOo2KDl87MUfPardImnfKxf9qJw-xZvLkZYl_BDRob1_4oYkE7h5TFqt11ba77ZCO5AXFsN7yp3P8ToAF5Fw |
| linkProvider | CAB International |
| openUrl | ctx_ver=Z39.88-2004&ctx_enc=info%3Aofi%2Fenc%3AUTF-8&rfr_id=info%3Asid%2Fsummon.serialssolutions.com&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.genre=article&rft.atitle=%E4%BA%8C%E6%96%91%E5%8F%B6%E8%9E%A8%E5%87%A0%E4%B8%81%E8%B4%A8%E9%85%B6%E5%9F%BA%E5%9B%A0%E7%9A%84%E5%8E%9F%E6%A0%B8%E8%A1%A8%E8%BE%BE%E5%8F%8A%E5%A4%9A%E5%85%8B%E9%9A%86%E6%8A%97%E4%BD%93%E5%88%B6%E5%A4%87&rft.jtitle=%E4%B8%9C%E5%8C%97%E5%86%9C%E4%B8%9A%E5%A4%A7%E5%AD%A6%E5%AD%A6%E6%8A%A5&rft.au=%E5%BC%A0%E9%81%93%E4%BC%9F+%E9%99%88%E9%9D%99+%E5%BC%A0%E6%AD%A3%E7%8E%B2+%E6%9B%BE%E7%87%95%E7%8E%B2+%E9%83%AD%E7%8E%89%E5%8F%8C&rft.date=2015&rft.issn=1005-9369&rft.volume=46&rft.issue=1&rft.spage=62&rft.epage=67&rft.externalDocID=663884911 |
| thumbnail_s | http://utb.summon.serialssolutions.com/2.0.0/image/custom?url=http%3A%2F%2Fimage.cqvip.com%2Fvip1000%2Fqk%2F90164A%2F90164A.jpg http://utb.summon.serialssolutions.com/2.0.0/image/custom?url=http%3A%2F%2Fwww.wanfangdata.com.cn%2Fimages%2FPeriodicalImages%2Fdbnydxxb%2Fdbnydxxb.jpg |