华北农学报
華北農學報
화북농학보
ACTA AGRICULTURAE BOREALI-SINICA
2014年
5期
39-44
,共6页
阚威%王华%马友记%赵兴绪%张勇
闞威%王華%馬友記%趙興緒%張勇
감위%왕화%마우기%조흥서%장용
奶牛隐性乳房炎%无乳链球菌%cfb基因%真核载体构建
奶牛隱性乳房炎%無乳鏈毬菌%cfb基因%真覈載體構建
내우은성유방염%무유련구균%cfb기인%진핵재체구건
Recessive bovine mastitis%Streptococcus agalactiaee%cfb gene%Eukaryotic expression vector
为研制奶牛乳房炎无乳链球菌的基因工程疫苗,采集隐性奶牛乳房炎奶样,利用THB ( Todd-Hewitt Broth )固体选择培养基和色素试验,并结合无乳链球菌种属特异性基因cfb,采用PCR技术从隐性乳房炎奶样中分离和鉴定无乳链球菌。根据GenBank已报道的链球菌cfb基因序列,经ORF分析和B细胞表位预测,利用Primer 5.0软件设计cfb因子富含B细胞表位区域引物,添加酶切位点和真核表达元件,以分离株无乳链球菌的基因组为模板,PCR扩增cfb基因并连接T载体克隆测序,经测序确定无误后,连接pcDNATM 3.1 V5-His A( pcDNA-cfb)真核载体,转化DH5α感受态细胞,提取重组质粒进行酶切和测序鉴定。结果显示,从89份隐性奶牛乳房炎奶样中分离得到8株无乳链球菌,成功构建了真核表达载体( pcDNA-cfb)。 THB和色素试验初步筛选,可以作为快速分离无乳链球菌的一种方法,利用Bcepred和Lasergene-Protean软件对分离得到的无乳链球菌cfb基因序列分析,在所克隆的cfb基因序列内有多个优势抗原表位,因此,有望作为无乳链球菌基因工程疫苗研制的抗原靶标基因序列,为进一步研究其基因工程疫苗提供基础条件。
為研製奶牛乳房炎無乳鏈毬菌的基因工程疫苗,採集隱性奶牛乳房炎奶樣,利用THB ( Todd-Hewitt Broth )固體選擇培養基和色素試驗,併結閤無乳鏈毬菌種屬特異性基因cfb,採用PCR技術從隱性乳房炎奶樣中分離和鑒定無乳鏈毬菌。根據GenBank已報道的鏈毬菌cfb基因序列,經ORF分析和B細胞錶位預測,利用Primer 5.0軟件設計cfb因子富含B細胞錶位區域引物,添加酶切位點和真覈錶達元件,以分離株無乳鏈毬菌的基因組為模闆,PCR擴增cfb基因併連接T載體剋隆測序,經測序確定無誤後,連接pcDNATM 3.1 V5-His A( pcDNA-cfb)真覈載體,轉化DH5α感受態細胞,提取重組質粒進行酶切和測序鑒定。結果顯示,從89份隱性奶牛乳房炎奶樣中分離得到8株無乳鏈毬菌,成功構建瞭真覈錶達載體( pcDNA-cfb)。 THB和色素試驗初步篩選,可以作為快速分離無乳鏈毬菌的一種方法,利用Bcepred和Lasergene-Protean軟件對分離得到的無乳鏈毬菌cfb基因序列分析,在所剋隆的cfb基因序列內有多箇優勢抗原錶位,因此,有望作為無乳鏈毬菌基因工程疫苗研製的抗原靶標基因序列,為進一步研究其基因工程疫苗提供基礎條件。
위연제내우유방염무유련구균적기인공정역묘,채집은성내우유방염내양,이용THB ( Todd-Hewitt Broth )고체선택배양기화색소시험,병결합무유련구균충속특이성기인cfb,채용PCR기술종은성유방염내양중분리화감정무유련구균。근거GenBank이보도적련구균cfb기인서렬,경ORF분석화B세포표위예측,이용Primer 5.0연건설계cfb인자부함B세포표위구역인물,첨가매절위점화진핵표체원건,이분리주무유련구균적기인조위모판,PCR확증cfb기인병련접T재체극륭측서,경측서학정무오후,련접pcDNATM 3.1 V5-His A( pcDNA-cfb)진핵재체,전화DH5α감수태세포,제취중조질립진행매절화측서감정。결과현시,종89빈은성내우유방염내양중분리득도8주무유련구균,성공구건료진핵표체재체( pcDNA-cfb)。 THB화색소시험초보사선,가이작위쾌속분리무유련구균적일충방법,이용Bcepred화Lasergene-Protean연건대분리득도적무유련구균cfb기인서렬분석,재소극륭적cfb기인서렬내유다개우세항원표위,인차,유망작위무유련구균기인공정역묘연제적항원파표기인서렬,위진일보연구기기인공정역묘제공기출조건。
In order to develop genetic engineering subunit vaccine ,milk samples of cows with subclinic mastitis were collected .THB ( Todd-Hewitt Broth ) solid selective medium and pigment test were adopted , combined with species specific gene cfb to isolate and identify Streptococcus agalactiae.ORF and B cell epitope analysis were car-ried out according to the cfb gene sequences published in GenBank .Primers designed with Primer 5 in cfb′s B cell epitope enriched sequence and eukaryotic expression elements and restriction enzyme sites were added .Genome DNA of the isolates were extracted and served as PCR templates for cfb amplification,prior to link with the T-A clo-ning vector for sequencing.Correctly cloned cfb sequence were then linked to the pcDNATM 3.1 V5-His A(pcDNA-cfb) and transformed to DH5αcompetent cell for plasmid proliferation and sequencing .Results showed 8 strains of Streptococcus agalactiaee were characterized from 89 milk samples,and the pcDNA-cfb was successfully constructed . Primary selection by THB selective medium and pigment test could be used for quick isolation of Streptococcus aga-lactiae.Analysis of the cfb sequence by Bcepred and Lasergene-Protean software showed the cloned cfb sequence contained multiple dominant B cell epitope ,thus had the potential to serve as target gene sequence for genetic engi-neering subunit vaccine of Streptococcus agalactiae.This work could served as the basis for further developing genet-ic engineering subunit vaccine for Streptococcus agalactiae.