两种AH100-ATS高压均质机在汉逊酵母细胞破碎中适用性的研究  

Study on the applicability of two AH100-ATS high-pressure homogenizers in cell disruption of Hansenula Polymorpha

作　　者：王玢 邵志伟 杨林鹏 陈卓涛 马素娟 杨千苇 周静 李薇 杨俊杰 周晖国 WANG Bin;SHAO Zhiwei;YANG linpeng;CHEN Zhuotao;MA Sujuan;YANG Qianwei;ZHOU Jing;LI Wei;YANG Junjie;ZHOU Huiguo(Lanzhou Institute of Biological Products Co.,Ltd.,Center for Gansu Provincial Vaccine Engineering Research,Lanzhou730046,Gansu Province,China)

机构地区：[1]兰州生物制品研究所有限责任公司,甘肃省疫苗工程技术研究中心,甘肃兰州730046

出　　处：《微生物学免疫学进展》2024年第1期48-54,共7页Progress In Microbiology and Immunology

基　　金：甘肃省科技重大专项(17ZD2FA007)。

摘　　要：目的 评价150型AH100-ATS高压均质机和40型AH100-ATS高压均质机在汉逊酵母细胞破碎中的适用性。方法 用2种AH100-ATS高压均质机分别重复破碎3组汉逊酵母,每组细胞破碎3次,光学显微镜下观察并比较2种高压均质机破碎汉逊酵母细胞前后的形态;SDS-PAGE电泳检测破碎后细胞中目的蛋白的释出;血球计数板计数且计算破碎后的细胞破碎率;Bradford法检测及计算蛋白释出度;计时破碎所需要的时间。结果 光学显微镜下,2种高压均质机破碎汉逊酵母细胞前后的细胞形态均可见明显差别;150型AH100-ATS高压均质机破碎汉逊酵母细胞后释出蛋白的粒径分布体积比高于40型AH100-ATS高压均质机,差异有统计学意义(P<0.05);SDS-PAGE电泳结果显示,150型AH100-ATS高压均质机破碎汉逊酵母后目的蛋白的灰度值高于40型AH100-ATS高压均质机,差异有统计学意义(P<0.05)。150型AH100-ATS高压均质机破碎汉逊酵母细胞3次后,细胞破碎率分别为24.16%、35.63%、48.67%,总细胞破碎率为65.70%;蛋白释出度分别为41.69%、38.29%、15.80%,破碎完成后总蛋白释出度为62.63%;平均使用时间分别为16.7、16.7、19.0 min,破碎完成后总时间为52.3 min。40型AH100-ATS高压均质机破碎破碎汉逊酵母细胞后细胞破碎率分别为16.10%、11.40%、28.78%,总细胞破碎率为58.41%;蛋白释出度分别为24.72%、21.04%、9.57%,破碎完成后总蛋白释出度为55.48%;平均使用时间分别为61.0、59.7、60.7 min,破碎完成后总时间为181.3 min。每次破碎的细胞破碎率、蛋白释出度、所需时间,以及总细胞破碎率、总蛋白释出度、总破碎时间,2种高压均质机差异均有统计学意义(P均<0.05)。结论 150型AH100-ATS破碎汉逊酵母的释出蛋白的粒径分布体积比、目的蛋白、细胞破碎率和蛋白释出度均高于40型AH100-ATS,150型AH100-ATS破碎所需的时间远少于40型AH100-ATS。150型AH100-ATS高压均质机可以用于中试大规模�Objective To evaluate the applicability of two AH100-ATS high-pressure homogenizers in Hansenula polymorpha cell fragmentation. Methods Two types of high-pressure homogenizers were used to repeatedly disrupt three groups of Hansenula polymorpha cells, each group of cells was broken three times, and the morphology of Hansenula polymorpha cells before and after being disrupted by the two types of high-pressure homogenizers were observed and compared under optical microscope. SDS-PAGE electrophoresis was used to detect the release of target proteins in fragmented cells. Fragmentation rate was calculated through counting cells before and after fragmentation. Count the blood cell count board and calculate the cell fragmentation rate. Bradford method was adopted for detecting and calculating protein release, as well optimizing the duration of bacterial fragmentation. Results Under the optical microscope, there were significant differences in the morphology of Hansenula polymorpha cells before and after fragmentation by two types of high-pressure homogenizers. However, no significant differences were observed in the morphology of cells after being disrupted by the two homogenizers. The particle size distribution of the released proteins of the type 150 AH100-ATS high-pressure homogenizer was higher than that of the type 40 AH100-ATS,with a statistically significant difference(P<0.05). SDS-PAGE electrophoresis results showed that Hansenula polymorpha cells disrupted by both types of high-pressure homogenizer released target proteins. However, the amount of released target proteins showed a significant difference(P<0.05),type 150 AH100-ATS was higher than that in the type 40. After continuous crushing of Hansenula polymorpha cells by high-pressure homogenizer for 3 times, the cell fragmentation rates of the 150 type AH100-ATS were 24.16%,35.63%,and 48.67%,respectively, with a final cell fragmentation rate of 65.70%. The protein release rates were 41.69%,38.29%,and 15.8%,respectively, and the total protein release rate of

关 键 词：高压均质机 汉逊酵母细胞 高压均质破碎 细胞破碎率 蛋白释出度 破碎时间 

分 类 号：R378.14[医药卫生—病原生物学]