CAJ | 학술논문

采用盐析法、重组蛋白A(HiTrap rProteinA Sepharose)亲和层析法分离纯化草鱼血清中的IgM,并通过SDS-PAGE及Western-blot技术对纯化蛋白的部分特性进行分析比较并制备兔抗IgM抗血清.结果表明:33%硫酸铵溶液可以沉淀血清中大部分蛋白,但电泳条带仍较多,其中含有78 kD和28 kD的条带,因此仅可作为免疫球蛋白粗提的方法;而rProteinA亲和层析法所提蛋白则仅有上述重链(78 kD)和轻链(28 kD).Western-blot显示,鼠抗人Ig抗体可与78 kD及28 kD条带发生发应.rProteinA亲和法提纯蛋白的纯度较高,但含量较低.条带较淡,仅可作为实验室小量提纯草鱼IgM的有效方法.将提纯的蛋白免疫实验兔后可制得效价高达1:25600的兔抗鱼IgM血清,并测得血清蛋白总量和IgM含量分别为25.87 mg和4.5 mg,IgM占血清蛋白总量的17.39%.本实验所采用的蛋白A亲和层析法提取草鱼血清IgM可以方便、快捷地获得高纯度的产物,适合在实验室中纯化鱼类lgM.同时本研究所制备的兔抗草鱼IgM血清也为今后的相关研究工作打下基础.
채용염석법、중조단백A(HiTrap rProteinA Sepharose)친화층석법분리순화초어혈청중적IgM,병통과SDS-PAGE급Western-blot기술대순화단백적부분특성진행분석비교병제비토항IgM항혈청.결과표명:33%류산안용액가이침정혈청중대부분단백,단전영조대잉교다,기중함유78 kD화28 kD적조대,인차부가작위면역구단백조제적방법;이rProteinA친화층석법소제단백칙부유상술중련(78 kD)화경련(28 kD).Western-blot현시,서항인Ig항체가여78 kD급28 kD조대발생발응.rProteinA친화법제순단백적순도교고,단함량교저.조대교담,부가작위실험실소량제순초어IgM적유효방법.장제순적단백면역실험토후가제득효개고체1:25600적토항어IgM혈청,병측득혈청단백총량화IgM함량분별위25.87 mg화4.5 mg,IgM점혈청단백총량적17.39%.본실험소채용적단백A친화층석법제취초어혈청IgM가이방편、쾌첩지획득고순도적산물,괄합재실험실중순화어류lgM.동시본연구소제비적토항초어IgM혈청야위금후적상관연구공작타하기출.
Immunoglobulins are the primary humoral component of the acquired immune system. In order to know the ImmunoglobulinM (IgM) of grass carp (Cienoyharyngodoni dellus), two different isolating methods were applied to find the best way to purify serum IgM in the present study. The purity and molecular weight of the serum IgM were determined, and then tile rabbit polyclonal antisera were prepared with the purified IgM. In this study, blood was collected with a syringe from the caudal sinus of 30 fish, and allowed to clot at 20℃ for 20 min and at 4℃ for 30 min. Serum was obtained by centrifugation at 500 g to remove cells and at 15000 g to remove particulate matter, and stored frozen at -80℃. Firstly, 33% ammonium sulfate precipitation was used to purify the serum, the result showed that most proteins in grass carp were precipitated and could not get high purity IgM, so it could only be a crude method. At the same time,HiTrap rProteinA Sepharose affinity was performed at 10℃ to purify IgM. The partial characteristics of purified proteins were then analyzed and compared by SDS-PAGE and Western-blot. The results revealed that grass carp serum immunoglobulin purified by HiTrap rProtein A affinity chromatography had only two bands of 78 kD and 28 kD, the same two bands as those in serum. Western-blot analysis showed that the mouse anti-human Ig antibody can recognize bands of 78 kD and 28 kD. Sera anti-IgM of grass carp had been prepared by repeatedly immunized New Zealand rabbits with purified IgM. and the titers of anti-sera obtained up to 1:25600 examined by indirect ELISA. Total serum protein and the concentration of IgM of the normal sera of grass carp were determined by using the Coomassie blue dye binding method of Bradford and sandwich ELISA, the values being 25.87 mg/mL and 4.5 mg/mL, respectively. The percentage of IgM in the total serum protein was 17.39%. Measurements of IgM levels in serum from several fish species have been surveyed and the results point to a range between 0.25 and 23.5 mg/mL. The studies also pointed to considerable individual variations in serum IgM levels among fish, as occurs with other fish immune activities. These changes may be related to size and/or age, environmental conditions or disease status. It must be emphasized, therefore,that the serum IgM values obtained in the present work represent the mean of at least thirty healthy and non-immunized individuals of similar size kept under same conditions. In present work, the 13mg amount of purified IgM from 10 mL serum was not larger than other fish because the grass carp was not immunized with antigen such as BSA in the research.In conclude, the results indicated that protein A-sepharose affinity chromatography was feasible in purification of IgM of grass crap. The polyclonal antibody obtained could be used in correlative studies in future.