中国组织工程研究
中國組織工程研究
중국조직공정연구
Journal of Clinical Rehabilitative Tissue Engineering Research
2014年
34期
5490-5496
,共7页
卢华定%连礼熠%陈明伟%戴驭虎
盧華定%連禮熠%陳明偉%戴馭虎
로화정%련례습%진명위%대어호
生物材料%材料相容性%壳聚糖%壳聚糖酶基因%克隆%表达%酶活性%国家自然科学基金
生物材料%材料相容性%殼聚糖%殼聚糖酶基因%剋隆%錶達%酶活性%國傢自然科學基金
생물재료%재료상용성%각취당%각취당매기인%극륭%표체%매활성%국가자연과학기금
biocompatible materials%chitosan%genes
背景：壳聚糖酶是高效、特异降解壳聚糖的酶，因此高效稳定地表达具有较高活性的壳聚糖酶可有效提高壳聚糖介导的基因治疗效果。<br> 目的：构建一种可高效降解壳聚糖的壳聚糖酶基因，探讨其在大肠杆菌中的表达及影响其活性的主要因素。方法：根据GenBank公布的曲霉菌CJ22-326内切型壳聚糖酶基因序列信息(EU302818)，设计并合成23条重叠引物，PCR 法扩增壳聚糖酶基因片段，构建原核表达质粒 pET28a-His6-CSN，将其转化大肠杆菌，收集融合蛋白His6-CSN，检测融合蛋白的表达及酶活性，同时检测不同pH值及温度对壳聚糖酶活性的影响。结果与结论：Western-blot证实融合蛋白His6-CSN成功表达，十二烷基硫酸钠-聚丙烯酰胺凝胶电泳检测显示表达的融合蛋白相对分子质量约为29000，二硝基水杨酸比色法测定壳聚糖酶对壳聚糖的降解活性显著高于溶菌酶(P <0.05)，但低于灰色链霉菌壳聚糖酶(P <0.05)。壳聚糖酶的最适pH值和最适温度分别为6.0和50℃，当pH值在4.0-7.0、温度在30-50℃范围内具有较高的酶活性。
揹景：殼聚糖酶是高效、特異降解殼聚糖的酶，因此高效穩定地錶達具有較高活性的殼聚糖酶可有效提高殼聚糖介導的基因治療效果。<br> 目的：構建一種可高效降解殼聚糖的殼聚糖酶基因，探討其在大腸桿菌中的錶達及影響其活性的主要因素。方法：根據GenBank公佈的麯黴菌CJ22-326內切型殼聚糖酶基因序列信息(EU302818)，設計併閤成23條重疊引物，PCR 法擴增殼聚糖酶基因片段，構建原覈錶達質粒 pET28a-His6-CSN，將其轉化大腸桿菌，收集融閤蛋白His6-CSN，檢測融閤蛋白的錶達及酶活性，同時檢測不同pH值及溫度對殼聚糖酶活性的影響。結果與結論：Western-blot證實融閤蛋白His6-CSN成功錶達，十二烷基硫痠鈉-聚丙烯酰胺凝膠電泳檢測顯示錶達的融閤蛋白相對分子質量約為29000，二硝基水楊痠比色法測定殼聚糖酶對殼聚糖的降解活性顯著高于溶菌酶(P <0.05)，但低于灰色鏈黴菌殼聚糖酶(P <0.05)。殼聚糖酶的最適pH值和最適溫度分彆為6.0和50℃，噹pH值在4.0-7.0、溫度在30-50℃範圍內具有較高的酶活性。
배경：각취당매시고효、특이강해각취당적매，인차고효은정지표체구유교고활성적각취당매가유효제고각취당개도적기인치료효과。<br> 목적：구건일충가고효강해각취당적각취당매기인，탐토기재대장간균중적표체급영향기활성적주요인소。방법：근거GenBank공포적곡매균CJ22-326내절형각취당매기인서렬신식(EU302818)，설계병합성23조중첩인물，PCR 법확증각취당매기인편단，구건원핵표체질립 pET28a-His6-CSN，장기전화대장간균，수집융합단백His6-CSN，검측융합단백적표체급매활성，동시검측불동pH치급온도대각취당매활성적영향。결과여결론：Western-blot증실융합단백His6-CSN성공표체，십이완기류산납-취병희선알응효전영검측현시표체적융합단백상대분자질량약위29000，이초기수양산비색법측정각취당매대각취당적강해활성현저고우용균매(P <0.05)，단저우회색련매균각취당매(P <0.05)。각취당매적최괄pH치화최괄온도분별위6.0화50℃，당pH치재4.0-7.0、온도재30-50℃범위내구유교고적매활성。
BACKGROUND:Chitosanase is an enzyme for efficient and special hydrolysis of chitoan, and hence its effective and stable expression with enzymatic activity wil contribute to improving gene therapeutic effect. <br> OBJECTIVE: To construct a chitosanase gene for the efficient and specifical hydrolysis of chitosan, and to investigate its expression inEscherichia coli and the main influencing factors of enzymatic activity. <br> METHODS:According to the sequences of endo-chitosanase ofAspergilus sp. CJ22-326 provided in Genbank (EU302818), primers were designed and synthesized. The Asperguilus endo-chitosanase gene was amplified by successive extension PCR. And then the recombinant pET28a-His6-CSN was constructed and expressed in <br> Escherichia coli BL21. Finaly the recombinant His6-CSN fusion protein was analyzed by sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE), the western blot and dinitrosalicylic acid assay for detecting the enzyme activity of eluted His6-CSN fusion protein. The influence of different pH value and temperature on the enzyme activity of the recombinant chitosanase was investigated. <br> RESULTS AND CONCLUSION: SDS-PAGE showed that 29 kDa proteins were expressed and the western blot assay showed that His6-CSN expressed successfuly in the host. Dinitrosalicylic acid assay determined the <br> enzymatic activity of His6-CSN was significantly higher than that of lysozyme, but lower than that of chitosanase from Streptomyces griseus (P < 0.05). The recombinant chitosanase displayed the maximal activity at temperature of 50℃ and pH value of 6.0. There were a higher enzymatic activity remaining at pH value of 4.0-7.0 and <br> temperature of 30-50℃.
