3株不同表型马尔尼菲青霉外分泌蛋白酶活性比较

中国真菌学杂志　 2014, Vol. 9 　Issue (4): 193-198.

论著下一篇

3株不同表型马尔尼菲青霉外分泌蛋白酶活性比较

王鹏¹, 冉玉平², 尹斌³, 庄凯文², 林新瑜⁴, 代亚玲⁵

1. 深圳市南山区人民医院皮肤性病科, 深圳 518052;
2. 四川大学华西医院皮肤性病科, 成都 610041;
3. 成都市第二人民医院皮肤性病科, 成都 610021;
4. 四川省人民医院皮肤性病科, 成都 610072;
5. 四川大学华西医院实验医学科微生物室, 成都 610041

收稿日期:2014-04-02 出版日期:2014-08-28 发布日期:2014-08-28
通讯作者: 冉玉平，E-mail：ranyuping@vip.sina.com E-mail:ranyuping@vip.sina.com
作者简介:王鹏，男（汉族），博士，副主任医师.E-mail：wangpeng02@126.com
基金资助:
国家自然科学基金（30570095）

Compare extracellular proteinase activity of three phynotypes of Penicillium marneffei

WANG Peng¹, RAN Yu-ping², YIN Bin³, ZHUANG Kai-wen², LIN Xin-yu⁴, DAI Ya-ling⁵

1. Department of Dermatology, Shenzhen Nanshan People's Hospital, Shenzhen 518052;
2. Department of Dermatology, West China Hospital, Sichuan University, Chengdu 610041;
3. Department of Dermatology, The Secend People's Hospital of Chengdu, Chengdu 610021;
4. Department of Dermatology, Sichuan Provincial People's Hospital, Chengdu 610072;
5. Department of Laboratory Medicine, West China Hospital, Sichuan University, Chengdu 610041

Received:2014-04-02 Online:2014-08-28 Published:2014-08-28

摘要/Abstract

摘要： 目的探讨如何有效诱导马尔尼菲青霉蛋白酶分泌及比较3株不同表型的马尔尼菲青霉菌株之间外分泌蛋白酶活性差异。方法选用临床分离的马尔尼菲青霉B-6323株（P株）及其两个不同表型的突变株M/M株和M/Y株，用以牛血红蛋白为底物的酵母碳基琼脂培养基（蛋白酶诱导培养琼脂基）诱导其外分泌蛋白酶分泌。将pH值为4.0、5.6及7.2的蛋白酶诱导培养琼脂基平板打孔后分别等量接种上述3株菌，分别置于25℃及37℃培养6 d后，行考马斯亮兰G-250染色，测定各菌株周围的透亮圈（蛋白分解环）直径。结果以牛血红蛋白为底物的酵母碳基琼脂培养基可有效诱导马尔尼菲青霉蛋白酶分泌;3株马尔尼菲青霉在任意3种pH值培养基中培养，37℃培养下菌周透亮圈直径大于25℃培养下，差异经t检验有统计学意义。经方差分析，37℃，pH4.0、5.6、7.2培养条件下马尔尼菲青霉菌周透亮圈直径之间差异有统计学意义（F值为97.198，P=0.000 1），用SNK法进行两两比较发现pH4.0与pH7.2菌周透亮圈直径及pH5.6与pH7.2菌周透亮圈直径差异均有统计学意义。当温度为37℃或25℃时，在任意一个pH值下，均为M/M株的菌周透亮圈直径均数最大，其次为P株，M/Y株最小。结论马尔尼菲青霉在37℃，酸性培养条件（pH4.0、5.6）下外分泌蛋白酶活性大，且不同表型株之间外分泌蛋白酶活性有差异，M/M株及P株为高外分泌蛋白酶活性株， M/Y株为低外分泌蛋白酶活性株。

关键词: 马尔尼菲青霉, 外分泌蛋白酶, 活性

Abstract: Objective To find the effective way to induce Penicillium marneffei (PM) to secrete extracellular proteinase and compare the extracellular proteolytic activity of three phynotypes of PM.Methods PM phynotypes (M/M and M/Y) and their parent strain B-6323 (P) were respectively inoculated with same amount into separate yeast carbon base (YCB) agar plates containing bovine hemoglobin (BHG) as the proteolytic substrate which had been adjusted to pH 4.0, pH 5.6, pH 7.2, and incubated at 25℃ and 37℃ for 6 days. The plates were stained with Coomassie blue G-250 to be observed clearly. Clear zones surrounding each clone result from broken down hemoglobin were measured to ascertain whether YCB-BHG agar can induce PM producing proteinase successfully, and to determine the best suitable condition for PM proteinase induced and compared the proteinase activity produced by the three isolates.Results YCB-BHG agar can successfully induce PM producing proteinase. Any three phynotypes of PM inoculated into medium at any three value of pH, the diameter of clear zones surrounded each clone increased greatly at 37℃ compared with at 25℃. It had statistically significant difference by t-test; The variance analysis showed that the diameters of clear zones surrounding each clone inoculated at pH4.0, 5.6, 7.2 medium at 37℃ had statistically significant difference (F-Values 97.198,P=0.0001). Student-Newman-Keuls (SNK) method was used to compare one to another. The diameters of clear zones surrounded the clone inoculated at pH4.0, pH5.6 medium and the clone inoculated at pH7.2 medium had statistically significant difference; No matter the culture temperature was 37℃ or 25℃, in any pH medium group, the M/M strain had greatest mean diameter of clear zone, then P strain, M/Y strain was lowest.Conclusions Extracellular proteinase of Penicillium marneffei growed at 37℃ in acidic medium (pH4.0 or pH5.6) showed greater proteolytic activity. The proteolytic activity produced by different morphotypes of Penicillium marneffei was different. The extracellular proteolytic activity of M/M strain and P strain was greater than M/Y strain after growth on the same condition.

Key words: Penicillium marneffei, extracellular proteinase, activity

中图分类号:

R379.9

王鹏, 冉玉平, 尹斌, 庄凯文, 林新瑜, 代亚玲. 3株不同表型马尔尼菲青霉外分泌蛋白酶活性比较[J]. 中国真菌学杂志, 2014, 9(4): 193-198.

WANG Peng, RAN Yu-ping, YIN Bin, ZHUANG Kai-wen, LIN Xin-yu, DAI Ya-ling. Compare extracellular proteinase activity of three phynotypes of Penicillium marneffei[J]. Chinese Journal of Mycology, 2014, 9(4): 193-198.

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3株不同表型马尔尼菲青霉外分泌蛋白酶活性比较

Compare extracellular proteinase activity of three phynotypes of Penicillium marneffei

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