Shipin Kexue (Jul 2024)

Gene Cloning and Prokaryotic Expression of the Allergen Tropomyosin from Macrobrachium nipponense

  • LUO Yeqing, ZHENG Shuangyan, SUN Yaobin, CHEN Jiao, LIU Xin, CHEN Hongbing, XIE Yanhai

DOI
https://doi.org/10.7506/spkx1002-6630-20230904-025
Journal volume & issue
Vol. 45, no. 13
pp. 89 – 95

Abstract

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To explore substitutes for natural tropomyosin as basic materials for the diagnosis and treatment of shrimp allergy, total RNA was extracted from Macrobrachium nipponense, and the full-length sequence of the tropomyosin gene was cloned by rapid amplification of cDNA ends (RACE). Specific primers were designed based on the cDNA sequence of tropomyosin and the expression plasmid pET-30a-TM was constructed. Finally, recombinant tropomyosin was expressed under the induction of isopropyl-β-D-thiogalactopyranoside (IPTG). The results showed that the full-length cDNA sequence of tropomyosin was 1 658 bp in length (GenBank accession number: OP974621). Its open reading frame (ORF) was 855 bp in length, encoding 284 amino acids, with a predicted protein isoelectric point of 4.70 and a predicted molecular mass of 32.8 kDa. The highest expression was obtained after 4 h of induction with a final IPTG concentration of 1 mmol/L at 37 ℃. The recombinant tropomyosin mainly existed as a soluble form in the supernatant of cell lysate with a molecular mass of approximately 38 kDa.

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