Cloning and Expression of the Leukotoxin BSBSE Gene from Fusobacterium necrophorum

以牛源坏死梭杆菌FNn株为试材,根据GenBank上已发表的坏死梭杆菌AF312861标准菌株的lktA序列设计1对引物,利用PCR技术扩增出1 131 bp的坏死梭杆菌白细胞毒素BSBSE基因。将PCR产物插入pGEM-T Easy vector中,经双酶切鉴定正确后进行序列测定。分析表明该BSBSE序列与GenBank上已发表的坏死梭杆菌AF312861标准菌株的lktA序列的核苷酸同源性为99%,推导出的氨基酸序列同源性为98%。为研究BSBSE的免疫原性,构建了原核表达载体pMAL-p2X-BSBSE,用IPTG诱导在大肠杆菌中表达。结果表明,BSBSE基因在大肠杆菌中进行了高效特异性融合表达,融合蛋白分子量约为84.5×103,其中41.5×103为BS-BSE基因表达的蛋白质,43.0×103为MBP融合标签,Western-blotting检测表明该表达产物有免疫原性。According to the sequence of announced lktA gene in Fusobacterium necrophorum,a pair of primers were designed.The BSBSE gene was amplified by PCR.The product was cloned into pGEM-T Easy vector.When nucleotide sequence and deduced amino acid sequence were compared with homologous sequence of the FN AF312861 lktA of GenBank,the homologue of the mucleotide sequence is 99% and the homologue of the amino acid sequence is 98%.The BSBSE fragment was inserted into expression vector pMAL-p2X and the plasmid pMAL-p2X-BSBSE were expressed in E.coli BL21 by IPTG induction.The SDS-PAGE analysis indicated the weight of the fusion protein was about 84.5.0×103,which included the 41.5×103 protein expressed from BSBSE gene and 43.0×103 fusion MBP tag.The recombinant BSBSE-pMAL-p2X production has Immunogenicity with western-blotting.The cloning and expression of the BSBSE gene established the foundation of further research on the function and application of the BSBSE gene.“十五”国家科技攻关子课题(2002BA518A04);; 中国农业科学院特产研究所科研基金项目(Tcs2005-03

Cloning and Expression of the Leukotoxin Gene SH from Fusobacterium necrophorum

坏死梭杆菌白细胞毒素是坏死杆菌病的主要致病因子,白细胞毒素基因(lkT)是其编码基因。以分离到的国内牛源坏死梭杆菌fn(A)菌株f4基因组dnA为模板,应用PCr方法扩增白细胞毒素基因SH片段,克隆至PMd18-T载体上,以bAMHⅠ和HIndⅢ酶切的目的片段SH与相应酶切的PET32A载体连接构建PET32A-SH重组表达质粒,经转化E COlI bl21(dE3)后用IPTg进行蛋白诱导,SdS-PAgE检测重组蛋白表达情况。结果表明:扩增基因序列大小为1800bP,SdS-PAgE检测重组蛋白有效表达,表达得到大小为80.2kdA的目的蛋白,采用镍柱亲和层析方法纯化SH重组蛋白,获得了纯度达95%的重组蛋白;经WEST-Ern-blOT证实,该蛋白对抗坏死杆菌阳性血清具有反应活性。The leukotoxin of Fusobacterium necrophorum(FN) is considered to be one of the main virulence factors.The lkt gene encodes for FN.In this study,the SH fragment of lkt gene was amplified by PCR using the F4 genome as the template,which was isolated from the Chinese Fusobacterium necrophorum strain.The fragment was then cloned to the pMD18-T vector for sequencing.Thereafter,the SH fragment was subcloned into the multiple cloning sites of the pET32 to construct pET32a-SH recombinant plasmid,which was then trans-formed into E.coli BL21(DE3) with IPTG induction for expression.SDS-PAGE was used to analyze the recombinant protein.The results showed that the SH fragment of about 1800 bp was amplified and was about 80.2 kDa.The fusion protein was purified by Ni-NTA affinity chromatography under denature conditions,and their purity was above 95%.Western-blot analysis indicated the SH fragment had anti-genicity against Fusobacterium necrophorum.“十五”国家科技攻关子课题(2002BA518A04);吉林省科技发展计划项目(20070570);吉林市科技发展计划项目(200805

Genetic Variation and Spread of Spartina alternifora in the Yellow River Delta

Spartina alternifora has been introduced into the Yellow River Delta since 1990.It spreads very rapidly and threatens the safety of the ecosystem.Investigating the genetic variation of Spartina alternifora can help understanding the mechanism of invasion and provide basic knowledge for the management of this invasive species.The genetic structure and diversity of four populations of Spartina alternifora(Pop 1,Pop 2,Pop 3 and Pop 4) at 13 loci were analyzed using microsatellite markers.The results revealed that the genetic diversity of Spartina alternifora in the Yellow River Delta was high.The average observed allele number(Na) was 7.385,the effective allele number(Ne) was 3.636,the Nei's genetic diversity was(0.700±0.094),the Shannon index was(1.453±0.343),and the proportion of polymorphic loci(P) was 100%.The Na,Ne,Nei's genetic diversity and Shannon index of Pop1 was the lowest,while those of Pop2-Pop4 were similar.However,the genetic differentiation(F_(st)=0.121) and the gene flow(Nm=1.825) among populations were medium.The genetic similarity among Pop 1-Pop 3 was high while the genetic similarity between Pop 4 and Pop 1-Pop 3 was low.As we known,Pop 3 was the initial and unique introduced population.The genetic structure of 4 populations suggested that firstly Pop 3 dispersed to Pop 4,and then Pop 3 dispersed to Pop 1 and Pop 2.The population dispersal order was not associated with the geographic distance of these populations,for the reason that the seeds of Spartina alternifora could disperse by sea water over a long distance.Therefore,developing environment-friendly herbicide to make Spartina alternifora in fecund could be a good way to control its invasion

JUNO Sensitivity on Proton Decay p→νˉK+p\to \bar\nu K^+ Searches

The Jiangmen Underground Neutrino Observatory (JUNO) is a large liquid scintillator detector designed to explore many topics in fundamental physics. In this paper, the potential on searching for proton decay in $p\to \bar\nu K^+$ mode with JUNO is investigated.The kaon and its decay particles feature a clear three-fold coincidence signature that results in a high efficiency for identification. Moreover, the excellent energy resolution of JUNO permits to suppress the sizable background caused by other delayed signals. Based on these advantages, the detection efficiency for the proton decay via $p\to \bar\nu K^+$ is 36.9% with a background level of 0.2 events after 10 years of data taking. The estimated sensitivity based on 200 kton-years exposure is $9.6 \times 10^{33}$ years, competitive with the current best limits on the proton lifetime in this channel

JUNO sensitivity on proton decay p → ν K + searches*

The Jiangmen Underground Neutrino Observatory (JUNO) is a large liquid scintillator detector designed to explore many topics in fundamental physics. In this study, the potential of searching for proton decay in the $p\to \bar{\nu} K^+$ mode with JUNO is investigated. The kaon and its decay particles feature a clear three-fold coincidence signature that results in a high efficiency for identification. Moreover, the excellent energy resolution of JUNO permits suppression of the sizable background caused by other delayed signals. Based on these advantages, the detection efficiency for the proton decay via $p\to \bar{\nu} K^+$ is 36.9% ± 4.9% with a background level of $0.2\pm 0.05({\rm syst})\pm$$0.2({\rm stat})$ events after 10 years of data collection. The estimated sensitivity based on 200 kton-years of exposure is $9.6 \times 10^{33}$ years, which is competitive with the current best limits on the proton lifetime in this channel and complements the use of different detection technologies