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Osmotic Regulation of Betaine Content in Leymus chinensis Under Saline-alkali Stress and cloning and expression of Betaine Aldehyde Dehydrogenase(BADH) Gene

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Chemical Research in Chinese Universities 2008年第2期24卷 204-209页

作者： CUI Xi-yan WANG Yong GUO Ji-xun Key Laboratory for Vegetation Ecology of Ministry of Education Institute of Grassland Science Northeast Normal University Changchun 130024 P. R. China School of Life Sciences Jilin Agricultural University Changchun 130118 P. R. China

The potted Leymus chinensis seedlings were treated with saline-alkali solution of six different（from Ⅰ to Ⅵ） concentrations. The results demonstrate that the betaine content and Betaine-aldehyde dehydrogenase（BADH： EC 1.***.1.8） activities have a direct relation with increased stressing time in the same treatment; both exhibit a single peak with increasing the concentration of saline-alkali solution, and number V shows the highest value. The BADH gene of Leyrnus chinensis was cloned by RT-PCR and RACE technology and was designated as LcBADH. The cDNA sequence of LcBADH was 1774bp including the open reading frame（ORF） of 1521bp（coding 506 amino acids）. The vector of prokaryotic expression was constructed by inserting the LcBADH gene fragmcnt into pET30a（＋） and transformed into E. coli BL21（DE3）. The result of SDS-PAGE shows that the idio-protein with a molecular mass of 56.78 kDa was effectively expressed in the recombinant bacteria induced by isopropyl fl-D-thiogalactoside（IPTG）.

关键词： Leymus chinensis Saline-alkali stress BADH RACE cloning and expression

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cloning and expression of the Tumstatin Active Peptides-T7 and its Derivant T7-NGR

Cloning and Expression of the Tumstatin Active Peptides-T7 a...

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第七届中国核学会“三核”论坛中国放射医学教育五十年暨中国毒理学会放射毒理委员会第八次全国会议

作者： HE Xin SONG Na-ling WANG De-zhi ZHAO Qi-ren Institute of Radiation Medicine Chinese Academy of Medical Sciences Peking Union Medical CollegeKey lab of TianJin Molecular Nuclear Medicine

Objective:To enhance the role targeting,design to link NGR sequence with tumstatin active peptides -T7’s C-terminal,the derivant called ***:The cloning vector pMD-T7 and pMD-T7N were constructed by PCR and gene synthesis methods respectively,identificated by digestion and DNA sequencing. After the digested plasmids were isolated by the low melting point agarose electrophoresis,the target-fragment was cut off and mixed with the recovery of the digested vector *** vector pET-T7 and pET-T7N were constructed in low melting point agarose,identificated by digestion and DNA sequencing,transformed into competent *** BL21(DE3),induced by ***:Identification result shows that pET-T7 and pET-T7N were ***-SDS-PAGE results showed that IPTG concentration of 1 mM,after the induction of 25℃,8h,T7 peptides and T7-NGR peptides have achieved the optimum conditions of ***: Has been successfully constructed the expression vectors of the two peptides,and got product,no coverage at home and abroad,laid the foundation for further activity experiments.

关键词： tumstatin T7 peptides T7-NGR guiding peptides cloning and expression

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cloning and expression of a bile salt hydrolase（bsh） gene from Lactobacillus plantarum

Cloning and expression of a bile salt hydrolase（bsh） gene ...

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第八届中国酶工程学术研讨会

作者： Zi-Xing Dong Juan Zhang Hua-Zhong Li Guo-Cheng Du Jian Chen Key Laboratory of Industrial Biotechnology Ministry of Educationand School of BiotechnologyJiangnan University State Key Laboratory of Food Science and Technology Jiangnan University

＜正＞Hypercholesterolemia is considered as a major risk for coronary heart disease *** lower price and less side effects than pharmacologic agents,oral administration of bile salt hydrolase（BSH）（EC 3.***.1.24）produced by probiotics can significantly reduce serum cholesterol *** this study,lactic acid bacteria with BSH activity were screened out by qualitative and quantitative ***

关键词： Hypercholesterolemia Bile salt hydrolase Lactobacillus plantarum cloning and expression

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cloning and Efficient expression of Bacillus sp. BH072 tas A Gene in Escherichia coli

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Transactions of Tianjin University 2015年第1期21卷 26-31页

作者：韩烨樊洁周志江檀茜倩赵鑫 School of Chemical Engineering and Technology Tianjin University

The Bacillus strain BH072 isolated from a honey sample showed strong antifungal activity against phytopathogen. Gene cloning test demonstrated that the strain had a tasA gene encoding an antifungal TasA protein. Although the wild strain simultaneously produced various antifungal substances, only the physicochemical property and antifungal activity of TasA protein were unclear due to the difficulty in extraction. In this study, tasA gene encoding the protein from Bacillus sp. BH072 was amplified by using the polymerase chain reaction （PCR） method and cloned into pET 28a （＋） vector, and then expressed in host cells Escherichia coli BL21 （DE3）. The expressed proteins were collected by centrifugation and ultrasonic treatment, and then purified by using nickel-nitrilotriacetic acid （Ni-NTA） metal affinity column and dialysis methods. The result of sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE） test showed that an expected protein band appeared with a size of 31 kDa. The expressed products possessed antifungal activity against the phytopathogenic indicator strain Botrytis cinerea. A genetically engineered strain tasA orE, coli was established in this study which can efficiently express Tas A protein.

关键词： Bacillus Tas A cloning and expression

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cloning of α-β fusion gene from Clostridium perfringens and its expression

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World Journal of Gastroenterology 2006年第8期12卷 1229-1234页

作者： Jia-Ning Bai Yan Zhang Bao-Hua Zhao College of Life Science Hebei Normal University Shijiazhuang 050016 HebeiProvince China

AIM： To study the cloning of α-β fusion gene from Closindium perfringens and the immunogenidty of 0-6 fusion expression. METHODS： cloning was accomplished after PCR amplification from strains NCTC64609 and C58-1 of the protective antigen genes of α-toxin and β-toxin. The fragment of the gene was cloned using plasmid pZCPAB. This fragment coded for the gene with the stable expression of α-β fusion gene binding. In order to verify the exact location of the α-β fusion gene, domain plasmids were constructed. The two genes were fused into expression vector pBV221. The expressed α-β fusion protein was identified by ELISA, SDS-PAGE, Western blotting and neutralization assay. RESULTS： The protective co-toxin gene （cpa906） and the β-toxin gene （cpb930） were obtained. The recombinant plasmid pZCPAB carrying α-β fusion gene was constructed and transformed into BL21（DE3）. The recombinant strain BL21（DE3）（pZCPAB） was obtained. After the recombinant strain BL21（DE3）（pZCPAB） was induced by 42℃, its expressed product was about 22.14% of total cellular protein at SDS-PAGE and thin-layer gel scanning analysis. Neutralization assay indicated that the antibody induced by immunization with α-βfusion protein could neutralize the toxicity of α-toxin and β-toxin. CONCLUSION： The obtained α-toxin and β-toxin genes are correct. The recombinant strain BL21（DE3）（pZCPAB） could produce α-β fusion protein. This protein can be used for immunization and is immunogenic. The antibody induced by immunization with α-β fusion protein could neutralize the toxicity of α-toxin and β-toxin.

关键词： Clostridium perfringens α-β fusion gene cloning and expression

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BmPLA2 containing conserved domain WD40 affects the metabolic functions of fat body tissue in silkworm, Bombyx moil

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Insect Science 2016年第1期23卷 28-36页

作者： Chabungbam Orville Singh Hu-Hu Xin Rui-Ting Chen Mei-Xian Wang Shuang Liang Yan Lu Zi-Zheng Cai Yun-Gen Miao Institute of Sericulture and Apiculture College of Animal Sciences Zhejiang University Hangzhou 310058 China

PLA2 enzyme hydrolyzes arachidonic acid, and other polyunsaturated fatty acids, from the sn-2 position to release free arachidonic acid and a lysophospholipid. Previous studies reported that the PLA2 in invertebrate organisms participates in lipid signaling molecules like arachidonic acid release in immune-associated tissues like hemo- cytes and fat bodies. In the present study, we cloned the BmPLA2 gene from fat body tissue of silkworm Bombyx mori, which has a total sequence of 1.031 kb with a 31.90 kDa pro- tein. In silico results of BmPLA2 indicated that the protein has a putative WD40 conserved domain and its phylogeny tree clustered with Danaus plexippus species. We investigated the transcriptional expression in development stages and tissues. The highest expression of BmPLA2 was screened in fat body among the studied tissues of third day fifth instar larva, with a high expression on third day fifth instar larva followed by a depression of expression in the wandering stage of the fifth instar larva. The expression of BmPLA2 in female pupa was higher than that of male pupa. Our RNAi-mediated gene silencing results showed highest reduction of BmPLA2 expression in post-24 h followed by post-48 and post-72 h. The BmPLA2-RNAi larvae and pupa could be characterized by pharate adult lethality and underdevelopment. The phenotypic characters of fat body cells in RNAi-induced larva im- plied that BmPLA2 affects the metabolic functions of fat body tissue in silkworm Bombyx mori.

关键词： BmPLA2 cloning and expression dsRNA interference fat body tissue silkworm

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