构建无抗性标记双拷贝透明质酸合成酶基因工程菌

Constructing duplication hasABC of chromosome recombinant in streptococcus equi subsp.zooepidemicu

摘要:

摘要:【目的】探讨一种构建无抗生素选择标记链球菌透明质酸合成酶基因工程菌的方法。【方法】PCR 扩增链球菌透明质酸合成酶操纵子hasABC 基因，用温度敏感载体pJR700 构建携带hasABC 基因重组质粒pXL32;电转化pXL32 质粒到马链球菌兽疫亚种感受态细胞，卡那霉素(Kanamycin，kan)平板37℃培养筛选重组子，在不含kan 液体培养基中30℃传代，37℃平板分离挑取抗生素敏感菌落，RT-PCR 检测工程菌染色体hasABC 基因表达，Bitter-Muir 法测定菌体透明质酸含量。【结果】在无抗生素选择压力条件下，获得透明质酸产率提高34% 的马链球菌兽疫亚种透明质酸合成酶基因工程菌。【结论】用pJR700 温度敏感载体系统，构建能提高透明质酸产量的无抗生素选择标记的基因工程菌是可行的。

Abstract:

Abstract:［Objective］To investigate the possibility of increasing the yield of hyaluronic acid by constructing duplication hasABC of chromosome recombinant in Streptococcus equi subsp． Zooepidemicus with a thermosensitive delivery vector system pJR700． ［Methods］We amplified a 4147 bp DNA fragment of hyaluronic acid synthase operon hasABC genes from chromatosome of S． zooepidemicus using PCR． This DNA fragment was subcloned into the pJR700 at ClaI sites to result in recombinant plasmid pXL32． The recombinant plasmid was transformed into S.Zooepidemicus by electroperation． The homologous recombination was induced by growing the bacteria at 37℃，and transformants were selected according to kanamycin resistance for 3 rounds． Then the culture was shifted to grow at 30℃ without antibiotics for 4 rounds to induce excision of the pJR700 indicated fragment． Colonies with kanamycin sensitivity were selected by plating on THY agar at 37℃．The hasABC recombinant of S． Zooepidemicus was identified through RT-PCR with primers homologous to the flanking regions．HA titers were measured by the modified carbazole assay． ［Results］We constructed successfully the duplication hasABC of chromosome recombinant of S.Zooepidemicus and the HA titer production by recombinants harboring duplication hasABC was 34% higher than that of the wild type at 24 h in shake flask culture.［Conclusion］The thermosensitive delivery vector of pJR700 could be used to construct the streptococcal hasABC recombinant strain for increasing the yield of HA in S. Zooepidemicus.