Li An-mei, Wu Ma-li, Huang Yu-ting, Guo Zhi-lai
Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. 2017 Feb;35(1):43-7.
To perform a proteomics analysis for metacercariae, juvenile and adult worms of Paragonimus skrjabini.
Crabs were collected in P. skrjabini endemic areas of Kaiyang and Bijie in Guizhou Province. Metacercariae were isolated, placed in PBS, and were used to infect three SD rats(10 metacercariae/rat) by intragastric administration and infect three male dogs(20 metacercariae/dog) through feeding. The rats were sacrificed at 1 month after infection to obtain juvenile worms. The dogs were sacrificed at 3 months after infection to obtain adult worms. The metacercariae, juvenile and adult worms were lysed, and total protein was extracted by ultrasonication. The total protein content was determined by Bradford method and separated by SDS-PAGE and two dimensional gel electrophoresis. Images of two dimensional gel electrophoresis were analyzed using the PDquest 8.0 software. The dots with difference were digested and analyzed with mass spectrometry. Finally, online searches in NCBI and local databases were performed.
Results of SDS-PAGE showed that the total protein of metacercariae, juvenile and adult worms was concentrated within Mr of 25 000-116 000. Fifty-one protein dots with difference were found by two dimensional gel electrophoresis, comprising of 20 dots for metacercariae, 25 for juvenile worms and 6 for adult worms. Thirty-six peptide sequences of metacercariae and juvenile worms were analyzed. They were basically determined to be Achrornobacter lyticus protease Ⅰ(a lysine-specific serine protease), ascorbate reductase protein, glutathione s-transferase DHAR2 analogue, heat shock proteins Hsp82 and Hsp96-β, actin, cystatin, etc., by online searches, and cysteine, actin and heat shock protein by local searches in the diginea database(downloaded to a local computer from NCBI). Mass spectrometry was not performed for adult worms, as the variation of grayscale value between their spots was far less than those for metacercariae and juvenile worms.
One difference is that the metacercariae of P. skrjabini have actin, while the juvenile worms have detoxification proteins and stress proteins. But they both have hydrolases and cysteine enzymes.
对斯氏狸殖吸虫囊蚴、童虫和成虫进行蛋白质组学分析。
在贵州省开阳和毕节的斯氏狸殖吸虫流行区采集蟹类。分离囊蚴，置于磷酸盐缓冲液（PBS）中，通过灌胃感染3只SD大鼠（每只大鼠10个囊蚴），并通过喂食感染3只雄性犬（每只犬20个囊蚴）。感染后1个月处死大鼠以获取童虫。感染后3个月处死犬以获取成虫。将囊蚴、童虫和成虫进行裂解，通过超声处理提取总蛋白。采用考马斯亮蓝法测定总蛋白含量，并通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和双向凝胶电泳进行分离。使用PDquest 8.0软件分析双向凝胶电泳图像。对有差异的点进行消化并通过质谱分析。最后，在NCBI和本地数据库中进行在线搜索。
SDS-PAGE结果显示，囊蚴、童虫和成虫的总蛋白集中在相对分子质量（Mr）为25 000 - 116 000范围内。双向凝胶电泳发现51个有差异的蛋白点，其中囊蚴有20个，童虫有25个，成虫有6个。分析了囊蚴和童虫的36个肽序列。通过在线搜索，它们基本被确定为溶菌无色杆菌蛋白酶Ⅰ（一种赖氨酸特异性丝氨酸蛋白酶）、抗坏血酸还原酶蛋白、谷胱甘肽S-转移酶DHAR2类似物、热休克蛋白Hsp82和Hsp96-β、肌动蛋白、胱抑素等，通过在吸虫数据库（从NCBI下载到本地计算机）中的本地搜索确定为半胱氨酸、肌动蛋白和热休克蛋白。由于成虫斑点之间灰度值的变化远小于囊蚴和童虫，因此未对成虫进行质谱分析。
一个差异是斯氏狸殖吸虫囊蚴有肌动蛋白，而童虫有解毒蛋白和应激蛋白。但它们都有水解酶和半胱氨酸酶。
