生物工程學報 25 August 2009, 25(8):1267～1272
鏈球菌噬菌體裂解酶在大腸桿菌中的表達,、純化及活性檢測
陳蔚青1, 王曉楓2, 王普2, 張德勇1, 陳虹1, 柯薇1, 陸胤1, 張建芬1
1 浙江樹人大學生物與環(huán)境工程學院, 杭州 310015
2 浙江工業(yè)大學藥學院, 杭州 310032
摘 要: 噬菌體裂解酶是噬菌體產(chǎn)生的細胞壁水解酶, 通過水解宿主菌細胞壁使子代噬菌體釋放, 在體外能高效且特異性地殺死細菌,。本研究旨在克隆和表達鏈球菌噬菌體裂解酶PlyC, 并測定其生物學活性。利用PCR方法擴增PlyC的2條肽鏈PlyCA和PlyCB, 構(gòu)建表達載體pET-32a(+)-PlyCA和pET-32a(+)-PlyCB, 分別轉(zhuǎn)化至大腸桿菌BL21(DE3)中, 以0.7 mmol/L IPTG在30 oC誘導7 h實現(xiàn)了高效表達, SDS-PAGE分析表明PlyCA和PlyCB表達量均可達菌體總蛋白的30%以上,。采用Ni2+-NTA親和層析法純化目的蛋白, 其純度大于95%,。用透析復性方法得到目的產(chǎn)物重組鏈球菌噬菌體裂解酶PlyC, 以濁度法和平板計數(shù)法檢測其體外抗菌效果, 掃描電子顯微鏡觀察裂解酶作用前后細菌細胞形態(tài)變化。結(jié)果表明重組PlyC能特異性裂解化膿性鏈球菌(A組 -溶血性鏈球菌), 以4 g/mL濃度作用于OD600為0.56的菌液60 min后殺菌率達99.6%, 掃描電鏡觀察結(jié)果顯示該酶作用于菌體后, 鏈球菌細胞裂解, 呈碎片狀態(tài),。本研究為開發(fā)一種新型,、高效的鏈球菌感染疾病治療藥物打下了基礎(chǔ)。
關(guān)鍵詞: 噬菌體裂解酶, 鏈球菌, 表達, 純化, 活性
Expression, purification and characterization of bacteriophage lysin of Streptococcus in Escherichia coli
Weiqing Chen1, Xiaofeng Wang2, Pu Wang2, Deyong Zhang1, Hong Chen1, Wei Ke1, Yin Lu1,and Jianfen Zhang1
1 College of Biological and Environmental Engineering, Zhejiang Shuren University, Hangzhou 310015, China
2 College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou 310032, China
Abstract: Lysins are murein hydrolases produced by bacteriophage that act on the cell wall of host bacteria to release progeny phages. Research indicated that lysins could kill bacteria effectively and specifically in vitro. To prepare recombinant bacteriophage lysin of Streptococcus (PlyC) and analyze its biological activity, we obtained two genes of PlyC named PlyCA and PlyCB by PCR amplification and inserted them into pET-32a(+), then transformed the recombinant expression vectors pET-32a(+)-PlyCA and pET-32a(+)-PlyCB into E. coli BL21(DE3) respectively. After induction with 0.7 mmol/L IPTG at 30 oC for 7 h, PlyCA and PlyCB were successfully expressed, SDS-PAGE analysis determined that they all constituted above 30% of the total cell proteins. After Ni2+-NTA affinity chromatography, the purity was more than 95%. With the denaturation and protein refolding, we gained the recombinant PlyC. To determine its biological activity, we adopted turbidimetry and plate count method. Before and after lysin treatment, the cell morphology was studied by scanning electron microscopy (SEM). The results showed that the recombinant PlyC could specifically cleavage Streptococcus pyogenes (group A β-hemolytic streptococci). Under the incubation time of 60 min with 4 μg/mL PlyC in Streptococcus pyogenes dilution which OD600 was 0.56, the germicidal effect was up to 99.6%, while SEM observations showed that cell wall cracked and presented cell debris. This finding laid the foundation for the further study and achieving an effective treatment for streptococcal infection.
Keywords: bacteriophage lysin, Streptococcus, expression, purification, activity
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