环形泰勒虫喀什株感染牛淋巴细胞系的建立及其生物学特征的鉴定

Establishment and characterization in vitro culture of a Theileria annulata Kashi-infected bovine lymphocyte cell line

建立1株环形泰勒虫(Theileria annulata)感染的牛淋巴细胞系,分析其生物学特征,为环形泰勒虫病疫苗研发提供新的细胞系。用环形泰勒虫喀什株感染的小亚璃眼蜱成蜱叮咬健康黄牛,当动物体温升高,体表淋巴结肿大时,用显微镜检查肩前和股前淋巴结穿刺物涂片,出现裂殖体感染的淋巴细胞时,手术摘取淋巴结,无菌条件下进行淋巴细胞的分离培养;选择合适的培养条件对该细胞进行适应性传代培养,可稳定传代培养后,绘制细胞生长曲线,测定细胞倍增时间和细胞周期,测定细胞系环形泰勒虫18S rRNA基因序列,通过序列比对分析其亲缘关系。结果显示,建立了1株可传代培养的环形泰勒虫喀什株裂殖体感染的淋巴细胞系(TaKashi),接种5.0×10~5 mL^(-1)的细胞,经72 h培养周期,细胞浓度可达到(2.8～3.0)×10~6mL^(-1),细胞存活率维持在90%以上;细胞群体倍增时间为33.4 h。细胞周期分析显示,G1期占46.2%,G2期占10.4%,S1期占43.4%,TaKashi传50代后仍然保持着淋巴细胞的形态学特征。18S rRNA基因序列比对及抗原基因Tams1同源性分析显示,该虫株与我国不同省份流行的环形泰勒虫虫株的相似性在91.9%以上,并处于同一个分支。

To establish a Theileria annulata infected bovine lymphocyte cell line and to analyze its biological characteristics that provides the opportunity for the development of vaccine against tropical theileriosis,adult Hyalomma anatolicum anatolicum ticks infected with T.annulata Kashi strain were feed on yellow cattle.Meanwhile,the physical and microscopic parameters of animal like body temperature,lymph node enlargement,microscopic examination of blood smear,and pre-scapular lymph node biopsy smear were performed for the appearance of schizonts in the lymphocytes.After confirmation of schizonts in lymphocytes,lymph nodes of infected animal were dissected aseptically for cell separation.The 18 S r RNA gene sequence of the cytoplasmic T.annulata was determined for its confirmation.A lymphocyte cell line(Ta Kashi) infected by the Kashi strain of the T.annulata schizont was established.After the stable in-vitro cell culture,the growth curve was drawn for the calculation of the cell doubling time and cell cycle.Cells density of 5.0×10^5mL^-1 reached to(2.8-3.0)×10^6mL^-1 after 72 h after in vitro culture of transformed cells in complete cell culture medium with the cell survival rate of 90% as determined.Recorded cell population doubling time was 33.4 h,and cell cycle analysis showed that G1,G2 and S1 stages were 46.2%,10.4%,and 43.4%,respectively.The morphological characteristics of Ta Kashi transformed lymphocytes remained unchanged until the 50 th generation.The comparative analysis of 18 S r RNA gene sequences and homogenous comparison of Tams1 gene of Ta Kashi strain showed that the similarity index between this strain and the other strains prevalent in different provinces of China were more than 91.9% and phylogenetic relationships were shown in the T.annulata branch.