Agriculture Department Key Laboratory of Mariculture & Stock Enhancement in North China's Sea, Dalian Key Laboratory of Marine Animal Disease Control and Prevention, College of Fisheries and Life Science, Dalian Ocean University, Dalian, 116023, China.
Agriculture Department Key Laboratory of Mariculture & Stock Enhancement in North China's Sea, Dalian Key Laboratory of Marine Animal Disease Control and Prevention, College of Fisheries and Life Science, Dalian Ocean University, Dalian, 116023, China.
Fish Shellfish Immunol. 2020 Mar;98:962-970. doi: 10.1016/j.fsi.2019.11.062. Epub 2019 Nov 26.
Siganus oraminl-amino acid oxidase (SR-LAAO), isolated from the serum of Siganus oramin, is an innate immune protein with significant antibacterial activity. The aim of this study was to express SR-LAAO in insect cells using a baculovirus expression system and evaluate the function of the recombinant SR-LAAO. To this end, an optimized sequence of the SR-LAAO gene was designed and synthesized, based on the codon bias of insect cells. Bacmid shuttle vectors and recombinant baculovirus were successfully constructed, and the recombinant baculovirus was transfected into Sf9 insect cells. The antibacterial activity and enzymatic characteristics of the recombinant SR-LAAO were investigated. The results showed that the pFastBac-optiSR-LAAO shuttle vectors and Bacmid-optiSR-LAAO were correctly constructed. The Sf9 insect cells exhibited significant cytopathic effects following infection with Bacmid-optiSR-LAAO and Bacmid; the specific PCR analysis proved that the recombinant baculovirus was successfully constructed. The immunofluorescence assay revealed that the recombinant baculovirus rSR-LAAO was abundantly expressed in infected Sf9 insect cells; the results of SDS/PAGE and Western blot analyses showed that a specific band appeared at about 60 kDa. Moreover, the crude rSR-LAAO enzyme displayed strong antibacterial activity against aquatic pathogens, particularly Streptococcus agalactiae and Streptococcus iniae. In addition, the results of catalase interference test implied that the antibacterial activity of rSR-LAAO was directly associated with (HO production). The results of the rSR-LAAO enzymatic characteristics test indicated that the Km value with l-Lysine as a substrate was 16.61 mM when the temperature was under 37 °C, and the optimum pH was 7. The antibacterial activity of rSR-LAAO could be completely inhibited by 10 mg/mL of pepsin, trypsin, and proteinase K compared with both methanol and acetone. Adding an equal volume of ethanol had a minimal impact on the antibacterial activity of rSR-LAAO. The crude enzyme could maintain a high level of antibacterial activity against both Gram-positive and -negative bacteria from 4 °C to 30 °C. In the present study, SR-LAAO was successfully expressed in Sf9 cells using the baculovirus expression system, and provides basic references for further research into the role of LAAO in marine animals and the development of new antimicrobial drugs.
从尖吻鲈血清中分离得到的 Siganus oramin-氨基酸氧化酶（SR-LAAO）是一种具有显著抗菌活性的先天免疫蛋白。本研究旨在利用杆状病毒表达系统在昆虫细胞中表达 SR-LAAO，并评估重组 SR-LAAO 的功能。为此，根据昆虫细胞的密码子偏好性，设计并合成了 SR-LAAO 基因的优化序列。成功构建了 bacmid 穿梭载体和重组杆状病毒，并将重组杆状病毒转染 Sf9 昆虫细胞。研究了重组 SR-LAAO 的抗菌活性和酶学特性。结果表明，成功构建了 pFastBac-optiSR-LAAO 穿梭载体和 Bacmid-optiSR-LAAO。Bacmid-optiSR-LAAO 感染 Sf9 昆虫细胞后，细胞出现明显的病变效应；特异性 PCR 分析证明重组杆状病毒构建成功。免疫荧光分析显示，重组杆状病毒 rSR-LAAO 在感染 Sf9 昆虫细胞中大量表达；SDS/PAGE 和 Western blot 分析结果表明，约 60 kDa 处出现特异性条带。此外，粗提 rSR-LAAO 酶对水产病原菌，特别是无乳链球菌和海豚链球菌具有较强的抗菌活性。此外，过氧化氢酶干扰试验的结果表明，rSR-LAAO 的抗菌活性与（HO 产生）直接相关。rSR-LAAO 酶学特性试验结果表明，当温度在 37°C 以下时，以 l-赖氨酸为底物时的 Km 值为 16.61 mM，最适 pH 值为 7。与甲醇和丙酮相比，10 mg/mL 的胃蛋白酶、胰蛋白酶和蛋白酶 K 可完全抑制 rSR-LAAO 的抗菌活性。与 rSR-LAAO 相比，加入等体积的乙醇对其抗菌活性的影响最小。粗酶在 4°C 至 30°C 之间对革兰氏阳性菌和革兰氏阴性菌均能保持较高的抗菌活性。本研究成功地利用杆状病毒表达系统在 Sf9 细胞中表达了 SR-LAAO，为进一步研究 LAAO 在海洋动物中的作用和开发新型抗菌药物提供了基础参考。
