应用改进的多克隆抗体Western blot技术研究柑桔速衰病毒蛋白

 本文利用多克隆抗体发展了一种无背景的Western blot技术并用于研究柑桔速衰病毒(CTV)的蛋白。结果表明,利用CTV兔多克隆抗体1212和1052发展的Western blot技术可以检测到感染CTV的墨西哥酸橙或Citrus excelsa植株内CTV的4种蛋白P1、P2、P3和P4。在健株或病株内杂交反应均无非特异性背景。从不同寄主上分离到的CTV不同分离物的蛋白条带是不同的。利用1212和1052抗体均可以检测到感染6个CTV分离物的墨西哥酸橙幼苗内的P1、P2和P3。利用1052抗体能检测到感染严重型分离物T36、T3和Mm2的墨西哥酸橙幼苗内微弱的P4,但感染轻型分离物T30、T26和T4的幼苗内则检测不到。利用1212抗体检测不到P4。在C. excelsa内,1212和1052抗体均能检测到感染所有分离物的病株内的P1。在感染T3、T26、T4或Mm2的病株内能检测到P2,但在感染T30和36分离物的病株内则检测不到。在感染T36、T3、T26、T4和Mm2的病株内可检测到P3,但在感染T30的病株内则检测不到。在大多数植物内,P1、P2、P3和P4的分子量分别约为25kDa,24kDa,21kDa和18kDa。在感染T36分离物的C. excelsa植株体内,P1和P3的分子量分别约为27kDa和22kDa,比感染其它分离物的C. excelsa和墨西哥酸橙内的P1和P3分子量略大。P1、P2和P3的分子量与利用单克隆抗体检测的CP,CP1和CP2的分子量相等。因此,P1、P2和P3可能是CTV的外壳蛋白CP,CP1和CP2。P4的特性不清楚。研究结果也表明,利用特异性的多克隆抗体进行的Western blot是研究CTV的一种有用的技术。应用该技术,病株内不同的CTV分离物或株系就可以通过特异性蛋白条带区分开来。

DEVELOPMENT OF WESTERN BLOT PROCEDURE FOR USING POLYCLONAL ANTIBODIES TO STUDY THE PROTEINS OF CITRUS TRISTEZA VIRUS

A non-back-ground reaction Western blot procedure for using polyclonal antibodies to study the proteins of citrus tristeza virus(CTV) was developed. The results showed four proteins, named P1, P2, P3 and P4, of CTV were detected in Mexican lime or Citrus excelsa plants that were infected with CTV by the Western blot procedure with CTV rabbit polyclonal antibodies, 1212 and 1052. No non-specific-back-ground reactions were detected in the healthy or diseased plants. The patterns of specific proteins of different isolates of CTV in different hosts were different. The P1, P2 and P3 proteins were observed in Mexican lime seedlings infected with 6 isolates of CTV by 1212 and 1052. Antibody 1052 detected a weak P4 band in blots from Mexican lime seedlings infected with the severe isolates, T36, T3 and Mm2, but not the mild isolates, T30, T26 and T4. Antibody 1212 does not detect P4. In Citrus excelsa , both the 1212 and 1052 antibodies detected P1 in trees infected with all isolates. P2 was detec ted in trees infected with T3, T26, T4 or Mm2, but not in trees infected with T30 and T36. P3 was detected in trees infected with T36, T3, T26, T4 and Mm2, but not T30. The molecular weights of P1, P2, P3, and P4 of CTV in most plants were about 25 kDa, 24 kDa, 21 kDa and 18 kDa, respectively. The molecular weights of P1 and P3 of T36 in Citrus excelsa plants were about 27 kDa and 22 kDa, respectively, a little larger than that of other isolates in both the Mexican lime and Citrus excelsa plants. Molecular weights of the P1, P2 and P3 were the same as those of CP, CP1 and CP2 detected by monoclonal antibodies. Therefore, the P1, P2 and P3 are probably the CTV coat proteins, CP, CP1 and CP2, respectively. The nature of P4 is unknown. The results also indicated that the Western blot procedure is a useful tool for studying CTV with CTV specific polyclonal antibodies. With the Western blot procedure, different isolates or strains of CTV could be distinguished by analysis of the specific protein patterns of the virus in infected host plants.