Howell M J, Hargreaves J J
Department of Zoology, Australian National University, Canberra.
Mol Biochem Parasitol. 1988 Feb;28(1):21-30. doi: 10.1016/0166-6851(88)90175-2.
Double stranded DNA complementary to poly(A)+ mRNA from the tapeworm Taenia ovis was cut with Sau 3A to an average length of about 300 bp and inserted into the Bam HI site of the expression plasmids pEX1, pEX2 and pEX3. These plasmids express a hybrid protein derived from a fusion of the cro gene with the lac Z gene (truncated at its 5' end by 53 bp) of phage lambda. Cloning sites lie downstream from the gene fusion. Escherichia coli infected with another plasmid (pCI857) bearing the temperature sensitive repressor of phage lambda was transformed with the pEX plasmids into which T. ovis DNA had been inserted; recombinants were selected by growth at 30 degrees C in the presence of ampicillin at 100 micrograms ml-1. Replicas were made and hybrid protein expression induced in recombinants by transferring them to 42 degrees C. Several recombinants expressing antigenic determinants of T. ovis were detected with T. ovis infected sheep serum that had been absorbed to remove antibodies to E. coli. Of five selected for further study, three expressed hybrid proteins of between 165 and 170 kDa of which the T. ovis component contributed between 48 and 55 kDa; in the other two, the tapeworm contribution was between 0.5 and 1.5 kDa. These antigenic determinants may be of some interest with respect to vaccine development since they are expressed during the normal course of T. ovis infection in sheep, and they are also present in the oncosphere - the infective larva of the parasite which stimulates immunity in sheep. The native antigens in adult worms and oncospheres that correspond to the antigenic determinants produced by the recombinant clones comprise a number of species ranging from 92.5 to 180 kDa. Tests with affinity purified antibodies indicate that the expressed products of the clones represent different epitopes on the same subset of polypeptides in both adult worms and oncospheres.
与绵羊带绦虫的聚腺苷酸加尾mRNA互补的双链DNA用Sau 3A切割成平均长度约为300bp，然后插入表达质粒pEX1、pEX2和pEX3的Bam HI位点。这些质粒表达一种杂合蛋白，该蛋白来源于噬菌体λ的cro基因与lac Z基因（在其5'端截短53bp）的融合。克隆位点位于基因融合的下游。用携带噬菌体λ温度敏感阻遏物的另一种质粒（pCI857）感染的大肠杆菌用已插入绵羊带绦虫DNA的pEX质粒进行转化；在含有100μg/ml氨苄青霉素的条件下于30℃培养以筛选重组体。制备复制品，并通过将重组体转移至42℃诱导杂合蛋白表达。用经吸收以去除抗大肠杆菌抗体的感染绵羊带绦虫血清检测到几个表达绵羊带绦虫抗原决定簇的重组体。在挑选出来作进一步研究的5个重组体中，3个表达165至170kDa之间的杂合蛋白，其中绵羊带绦虫成分占48至55kDa；在另外两个重组体中，绦虫成分在0.5至1.5kDa之间。这些抗原决定簇对于疫苗开发可能具有一定意义，因为它们在绵羊带绦虫感染绵羊的正常过程中表达，并且它们也存在于六钩蚴中——寄生虫的感染性幼虫，可刺激绵羊产生免疫。与重组克隆产生的抗原决定簇相对应的成虫和六钩蚴中的天然抗原包括多种分子量在92.5至180kDa之间的物种。用亲和纯化抗体进行的试验表明，克隆的表达产物代表成虫和六钩蚴中同一多肽亚群上的不同表位。
