水貂生长激素的原核表达及特异性抗血清制备

doi:10.16431/j.cnki.1671-7236.2018.04.004

Abstract

Abstract:

This study was aimed to establish a prokaryotic expression system of mink growth hormone (mGH),explore a scale production method,and get specific anti-mGH serum from the immune rabbit.In this study,the prokaryotic expression vector pET28b-mGH was transferred into Rosetta(DE3)^TM pLysS competent cells and the inclusion body protein was got successfully which induced by IPTG.The protein was dissolved and refolded with 8 mol/L urea solution,the recombinant protein was obtained by Ni-NTA resin affinity chromatography purification.The recombinant protein was confirmed by Western blotting and MALDI-TOF-MS methods,the purity and concentration of mGH fusion protein were determined by SDS-PAGE and BAC methods,respectively.In the case of refolding mGH protein,the immune antigen was prepared with a complete fluoride adjuvant or incomplete fluoride adjuvant.Three New Zealand White rabbits were immunized by subcutaneous multipoint injection the immune antigen,the first dose was 600 μg per rabbit,the second dose was 600 μg per rabbit,the third dose was 400 μg per rabbit.We took the rabbits' blood sampling on 10 day after the third immunization,and then separated antibody serum,the anti-mGH serum specificity and antibody titer were identified by Western blotting and indirect ELISA,respectively.The result showed that the purified protein was mGH fusion protein,SDS-PAGE results showed that the purity of the mGH fusion protein was more than 90%,and the BCA result showed that the concentration reached to 669 μg/mL.The rabbit anti mGH serum was specificity,the anti-mGH sera with a titer of over 1:256 000.The results showed that the prokaryotic expression vector pET28b-mGH could express efficiently mGH fusion protein in Rosetta (DE3)^TM pLysS cells,the expressed protein was immunized with New Zealand White rabbits after denaturation,renaturation and purification,which could be used to prepare high titer specific antiserum.

Key words: mink growth hormone (mGH); prokaryotic expression; Ni-NTA resin; mass spectrometry; rabbit anti-mGH serum

CLC Number:

Q786

SU Jun, LIU Man, WANG Jigui, SUN Yan, LIU Weiquan. Prokaryotic Expression and Specific Antiserum Preparation of Mink Growth Hormone[J]. , 2018, 45(4): 866-872.

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Prokaryotic Expression and Specific Antiserum Preparation of Mink Growth Hormone

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