凝结芽胞杆菌IPE22中乳酸脱氢酶基因的克隆表达与特征分析

Cloning Expression and Feature Analysis of Lactate dehydrogenase Gene in Bacillus coagulans IPE22

Bacillus coagulans IPE22是一株重要的乳酸生产菌,对其乳酸脱氢酶（lactate dehydrogenase,LDH）基因进行克隆、表达和生物信息学分析十分必要。以B.coagulans36D1序列信息（Gen Bank：CP003056.1）设计引物,PCR扩增获得B.coagulans IPE22中LDH（Bc-LDH）基因,构建重组表达质粒并转化Escherichia.coli BL21（DE3）中进行诱导表达,针对Bc-LDH的氨基酸序列进行生物信息学特征分析。经基因片段核酸电泳分析和SDS-PAGE电泳验证,Bc-LDH基因成功实现克隆表达。Bc-LDH为等电点5.41的亲水性蛋白质,具有多个糖基化和磷酸化位点,二级结构以不规则卷曲,α-螺旋和延伸链为主要结构元件。Bc-LDH具有NADB-Rossmann和Ldh-1-C超家族功能结构域,包含23个氨基酸组成的跨膜结构域,且不同乳酸生产菌中LDH均具有高度保守的活性位点。蛋白质相互作用预测显示,Bc-LDH与细胞内丙酮酸激酶和NAD结合苹果酸蛋白的互作最为密切。

B acillus coagul ans IPE22 is an important lactic acid producer. The cloning, expression and bioinformatics analysis of its lactate dehydrogenase（LDH） gene is very necessary. The primers were designed by B.coagulans 36 D1 sequence information（Gen Bank： CP003056.1）. The LDH（Bc-LDH） gene in B. coagulans IPE22 was amplified by PCR amplification. The recombinant expression plasmid was constructed and transformed into Escherichia coli BL21（DE3） to induce expression. Then, the bioinformatics characteristics of the amino acid sequences of Bc-LDH were analyzed. The results showed that the cloned gene of Bc-LDH was expressed successfully confirmed by DNA electrophoresis and SDS-PAGE electrophoresis. Bc-LDH is a hydrophilic protein with isoelectric point of 5.41. Several phosphorylation sites and glycosylation sites were founded in Bc-LDH. In the secondary structure, the atactic coil, α-helix and extended strand were main structural elements. Bc-LDH had a super family domain of Ldh-1-C and NADB-Rossmann, and contained a trans-membrane region formed by23 amino acids. LDH of different lactic acid producing bacteria all had highly conserved active sites. Results of protein interaction prediction showed that Bc-LDH interacted most closely with pyruvate kinase and malic proteinNAD-binding protein.