CAJ | 학술논문

背景:国外有1篇文献报道了肝再生增强因子在中枢神经系统内的分布与表达.由于关于重组大鼠肝再生增强因子蛋白多克隆抗体制备、鉴定及原核表达载体如何构建的相关文献较少,国内对于其在中枢神经系统的研究目前未见报道.目的:利用大肠杆菌BL21表达大鼠肝再生增强因子融合蛋白,并制备和鉴定其多克隆抗体.方法:提取SD大鼠海马组织RNA,构建原核表达重组质粒pET28a-ALR并转化到宿主菌BL21中,经异丙基-β-D-硫代半乳糖苷诱导表达目的融合蛋白,经Ni2+亲和层析纯化回收,4次免疫日本大耳白兔后,心脏取血,吸取血清作为多克隆抗体,以ELISA测定抗体血清效价,Western-blotting检测抗体的特异性和亲和性.观察:①原核表达重组质粒pET28a-ALR构建.②pET28a-ALR重组体酶切鉴定.③ELISA及Western-blotting检测结果.结果与结论:双酶切电泳检测结果显示得到了预期条带,其大小分别为5.3 kb和0,4 kb,经核苷酸序列分析证明成功构建了pET28a-ALR原核表达载体.成功获得分子质量约为19 ku纯化的融合蛋白.ELISA测定显示多克隆抗体效价可达到1:2 000,说明抗体与纯化的重组肝再生增强因子蛋白具有良好的反应性,有较高的效价,可满足实验的要求.通过Western-blotting检测证明该抗体可以特异性识别肝再生增强因子蛋白.实验成功地利用原核表达体系表达了肝再生增强因子融合蛋白,并制备、纯化了抗肝再生增强因子蛋白的多克隆抗体,经鉴定能够满足针对肝再生增强因子免疫印迹检测的实验要求.
배경:국외유1편문헌보도료간재생증강인자재중추신경계통내적분포여표체.유우관우중조대서간재생증강인자단백다극륭항체제비、감정급원핵표체재체여하구건적상관문헌교소,국내대우기재중추신경계통적연구목전미견보도.목적:이용대장간균BL21표체대서간재생증강인자융합단백,병제비화감정기다극륭항체.방법:제취SD대서해마조직RNA,구건원핵표체중조질립pET28a-ALR병전화도숙주균BL21중,경이병기-β-D-류대 반유당감유도표체목적융합단백,경Ni2+친화층석순화회수,4차면역일본대이백토후,심장취혈,흡취혈청작위다극륭항체,이ELISA측정항체혈청효개,Western-blotting검측항체적특이성화친화성.관찰:①원핵표체중조질립pET28a-ALR구건.②pET28a-ALR중조체매절감정.③ELISA급Western-blotting검측결과.결과여결론:쌍매절전영검측결과현시득도료예기조대,기대소분별위5.3 kb화0,4 kb,경핵감산서렬분석증명성공구건료pET28a-ALR원핵표체재체.성공획득분자질량약위19 ku순화적융합단백.ELISA측정현시다극륭항체효개가체도1:2 000,설명항체여순화적중조간재생증강인자단백구유량호적반응성,유교고적효개,가만족실험적요구.통과Western-blotting검측증명해항체가이특이성식별간재생증강인자단백.실험성공지이용원핵표체체계표체료간재생증강인자융합단백,병제비、순화료항간재생증강인자단백적다극륭항체,경감정능구만족침대간재생증강인자면역인적검측적실험요구.
BACKGROUND:An abroad study repoRed the distribution and expression of augmenter of liver regeneration(ALR)in the central nervous system.There are few literatures on how to prepare and evaluate ALR protein polyclonal antibody in recombinant rats,and how to construct prokaryotic expression vector.There are no repots concerning ALR in the central nervous system in China.OBJECTIVE:TO express ALR fusion protein in E coli BL21 and prepare and identify polyclonal antibody.METHODS:RNA was extracted from the hippocampus of Sprague Dawley rats.The prokaryotic expression plasmid pET28a-ALR was constructed and the positive recombinant plasmid was transformed into BL21.Protein ALR was expressed by inducing transformed BL21 with Isopropyl-β-D-thiogalactopyranoside(IPTG)and purified by Ni~(2+)affinity chromatography column after immune the rabbit for 4 times.the serum of rabbits was extracted from hear as polyclonal antibody.The titer and specificity of the rabbit's antiserum was respectively measured by ELISA and Western blotting The following parameters were measured:construction of prokaryotic expression plasmid pET26a-ALR;pET28a-ALR recombinant enzyme digestion evaluation;results of ELISA and Western-blotting.RESULTS AND CONCLUSION:Expecting bands were obtained by double enzyme digestion electrophoresis,respectively 5.3 kb and 0.4 kb.Nucleotide sequence analysis verified that prokaryotic expression vector pET28a-ALR was successfully constructed.The 19 ku fusion protein was successfuIly expressed.The titer of the antiserum measured by ELlSA could achieve 1:2 000 This indicated that antibody and purified recombinant ALR had a good reaction.and high titer.could meet the experimental require.Western blotting analysis proved that the antibody could identify the prokaryotic expression product of ALR.Prokaryotic expression system expressed ALR fusion protein,prepared and purified polyclonal antibody of ALR protein,and could meet the experimental require of ALR immunoblotting.