河北农业科学
河北農業科學
하북농업과학
JOURNAL OF HEBEI AGRICULTURAL SCIENCES
2014年
2期
52-59
,共8页
马志民%秦君%杨春燕%邸锐%闫龙%张梅申%李子千%李亚璞%史晓蕾%鲍聪%许宁%封树平%张孟臣
馬誌民%秦君%楊春燕%邸銳%閆龍%張梅申%李子韆%李亞璞%史曉蕾%鮑聰%許寧%封樹平%張孟臣
마지민%진군%양춘연%저예%염룡%장매신%리자천%리아박%사효뢰%포총%허저%봉수평%장맹신
脂肪氧化酶%同功酶%单克隆抗体%多克隆抗体%双抗体夹心法%联合鉴定
脂肪氧化酶%同功酶%單剋隆抗體%多剋隆抗體%雙抗體夾心法%聯閤鑒定
지방양화매%동공매%단극륭항체%다극륭항체%쌍항체협심법%연합감정
Lipoxygenase%Isoenzyme%Mono-clonal antibody%Polyclonal antibody%Double anti-body sandwich method%Joint identification
本研究目的是建立一套适宜于育种后代材料脂肪氧化酶缺失类型筛选鉴定的方法。首先选择适用于近等基因系材料的消减免疫法，以单缺Lox2的J12-2作为耐受原，将含有3种Loxs的Soybean Lox （ Type I-B）作为抗原，利用该方法筛选到1株能稳定分泌 Lox2单抗的杂交瘤细胞株 Lox2-1。在解决了 Lox2单抗的基础上，进一步以纯化的Lox1和Lox3作抗原，按常规方法，制备并筛选到1株能稳定分泌 Lox1单抗的杂交瘤细胞株Lox1-1，4株Lox3的杂交瘤细胞株Lox3-1、 Lox3-2、 Lox3-3和Lox3-4；利用腹水诱导法生产其细胞株抗体，腹水效价在1∶20000与1∶800000之间，经鉴定6株单抗均具有较强的特异性。以Soybean Lox （ TypeI-B ）作抗原制备兔多克隆抗体血清，其针对 Lox1、 Lox2和 Lox3的效价均达到1∶76800。最终建立了以兔多抗作固相抗体， Lox1-1、 Lox2-1、 Lox3-4分别作检测抗体的对大豆脂氧酶缺失类型进行联合鉴定的技术体系，该技术不但为无腥大豆育种提供了技术保障，还为其他同工酶检测技术的开发积累了宝贵经验。
本研究目的是建立一套適宜于育種後代材料脂肪氧化酶缺失類型篩選鑒定的方法。首先選擇適用于近等基因繫材料的消減免疫法，以單缺Lox2的J12-2作為耐受原，將含有3種Loxs的Soybean Lox （ Type I-B）作為抗原，利用該方法篩選到1株能穩定分泌 Lox2單抗的雜交瘤細胞株 Lox2-1。在解決瞭 Lox2單抗的基礎上，進一步以純化的Lox1和Lox3作抗原，按常規方法，製備併篩選到1株能穩定分泌 Lox1單抗的雜交瘤細胞株Lox1-1，4株Lox3的雜交瘤細胞株Lox3-1、 Lox3-2、 Lox3-3和Lox3-4；利用腹水誘導法生產其細胞株抗體，腹水效價在1∶20000與1∶800000之間，經鑒定6株單抗均具有較彊的特異性。以Soybean Lox （ TypeI-B ）作抗原製備兔多剋隆抗體血清，其針對 Lox1、 Lox2和 Lox3的效價均達到1∶76800。最終建立瞭以兔多抗作固相抗體， Lox1-1、 Lox2-1、 Lox3-4分彆作檢測抗體的對大豆脂氧酶缺失類型進行聯閤鑒定的技術體繫，該技術不但為無腥大豆育種提供瞭技術保障，還為其他同工酶檢測技術的開髮積纍瞭寶貴經驗。
본연구목적시건립일투괄의우육충후대재료지방양화매결실류형사선감정적방법。수선선택괄용우근등기인계재료적소감면역법，이단결Lox2적J12-2작위내수원，장함유3충Loxs적Soybean Lox （ Type I-B）작위항원，이용해방법사선도1주능은정분비 Lox2단항적잡교류세포주 Lox2-1。재해결료 Lox2단항적기출상，진일보이순화적Lox1화Lox3작항원，안상규방법，제비병사선도1주능은정분비 Lox1단항적잡교류세포주Lox1-1，4주Lox3적잡교류세포주Lox3-1、 Lox3-2、 Lox3-3화Lox3-4；이용복수유도법생산기세포주항체，복수효개재1∶20000여1∶800000지간，경감정6주단항균구유교강적특이성。이Soybean Lox （ TypeI-B ）작항원제비토다극륭항체혈청，기침대 Lox1、 Lox2화 Lox3적효개균체도1∶76800。최종건립료이토다항작고상항체， Lox1-1、 Lox2-1、 Lox3-4분별작검측항체적대대두지양매결실류형진행연합감정적기술체계，해기술불단위무성대두육충제공료기술보장，환위기타동공매검측기술적개발적루료보귀경험。
It was established in this study that applies to the missing type screening and identification of the soybean lipoxygenases for the breeding of the non-odor soybean.Selection the method which suitable for near isogenic lines of subtractive immunization, and using J12-2 missing Lox2 as the toleragen and Soybean Lox (Type I-B) containing Lox1 , Lox2 and Lox3 simultaneously as the antigen, and hybridomas secreting antibodies against Lox2 were selected and cloned.In the settlement of the foundation of Lox2 monoclonal antibody, the hybridoma cell lines secreting anti-Lox1 and anti-Lox3 McAbs respectively were obtained by immunizing the BALB/C mice with the soybean lipoxygenases purified by ourselves, and one stable McAbs Lox1-1 against Lox1, and four ( Lox3-1, Lox3-2, Lox3-3, Lox3-4 ) against Lox3.These hybridomas were prepared ascites of the mice, and the titers of the McAbs in the ascites ranged from 1∶20 000 to 1∶800 000, and these McAbs all had strong specificity.The rabbit polyclonal antibody in serum anti Lox1 , Lox2 and Lox3 simultaneously was obtained by immunizing the New Zealand white rabbits with Soybean Lox ( Type I-B) , and the titer all achieves 1∶76 800 that against Lox1, Lox2 and Lox3 respectively.In the end, the technical system for the the missing type screening and identification of the soybean lipoxygenases was established using the rabbit polyclonal antibody as the solid-phase antibody, and the McAbs Lox1-1, Lox2-1, Lox3-4 as the detectable antibodies respectively.It provided the technical support for the breeding of the non-odor soybean and accumulating valuable experience for developing the other isoenzyme detection technology.
