China Animal Husbandry and Veterinary Medicine ›› 2022, Vol. 49 ›› Issue (5): 1621-1629.doi: 10.16431/j.cnki.1671-7236.2022.05.003

• Biotechnology • Previous Articles     Next Articles

Prokaryotic Expression and Activity Identification of LbCas12a Protein

LIU Ru1,2, LI Xiaolong1,2, ZHANG Xiaoqian1,2, TIAN Xiaohuan1,2, YU Mei1,2,3, ZHAO Shuhong1,2,3, CAO Jianhua1,2,3   

  1. 1. Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education, Huazhong Agricultural University, Wuhan 430070, China;
    2. Key Laboratory of Swine Genetics and Breeding of Ministry of Agriculture and Rural Affairs, Huazhong Agricultural University, Wuhan 430070, China;
    3. The Cooperative Innovation Center for Sustainable Pig Production, Huazhong Agricultural University, Wuhan 430070, China
  • Received:2021-10-21 Online:2022-05-05 Published:2022-04-29

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Abstract: 【Objective】 The purpose of this study was to obtain LbCas12a protein from Lachnospiraceae ND2006 with in vitro cleavage activity,in order to provide an important biological tool for the application of LbCas12a.【Method】 LbCas12a gene was synthesized according to the gene sequence of pMBP-LbCas12a plasmid (Addgene,113431),and homologous recombination was performed with pET-28a(+) linearized vector.The recombinant plasmid pET28a-LbCas12a was constructed.After sequencing and double enzyme digestion,the positive recombinant plasmid was transferred into the BL21(DE3) for IPTG induced expression.The optimal concentration and temperature induced by IPTG were detected by 12% SDS-PAGE,and the expression form was identified.The protein concentration was detected by Ni-NTA resin affinity chromatography and ultrafiltration concentration,and the protein concentration was detected by BCA method.The concentrated LbCas12a was co-incubated with Porcine parvovirus (PPV) target DNA,CRISPR RNA (crRNA) and ssDNA reporter probe FQ,a control group without LbCas12a protein and four experimental groups with different protein concentration gradients (125,250,500,1 000 nmol/L) were set up.The fluorescence intensity of ssDNA probes in different groups was detected by automatic microplate reader,and the activity of PPV at the test site was detected.【Results】 Sequencing and NheⅠ and SalⅠ double enzyme digestion results showed that the recombinant plasmid pET28a-LbCas12a was successfully constructed.The results of 12% SDS-PAGE showed that the optimum induction concentration of IPTG was 0.5 mmol/L,the optimum induction temperature was 37 ℃,and the expression form was mainly soluble expression.The results of protein concentration detection showed that the concentration of protein was 485 ng/μL and the weight was 143 ku.The activity test results showed that the fluorescence intensity of 125,250,500,1 000 nmol/L LbCas12a protein was extremely significantly higher than that of control group (P<0.01).【Conclusion】 In this study,LbCas12a protein with high activity was successfully expressed,and LbCas12a protein had trans-cleavage activity of ssDNA in vitro,which laid a foundation for the subsequent molecular detection technology based on CRISPR-LbCas12a system.

Key words: LbCas12a protein; prokaryotic expression; protein purification; trans-activity

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