镰形扇头蜱两个组织蛋白酶L-样半胱氨酸蛋白酶新基因的克隆与序列分析

蜱是动物常见的外寄生虫，并且传播多种人和动物的疾病，严重危害畜牧业发展和人类健康。为了寻找基因工程疫苗候选抗原基因，根据半胱氨酸蛋白酶的保守性氨基酸序列及镰形扇头蜱氨基酸密码子偏好设计引物，PCR扩增、测序并分析得到2个镰形扇头蜱的半胱氨酸蛋白酶基因片段cysA和cysB，再通过RACE的方法得到全长基因序列。cysA全长168bp，编码332个氨基酸；cysB全长1153bp，编码335个氨基酸。经过分析, CysA和CysB均与其他蜱种或物种的组织蛋白酶L样半胱氨酸蛋白酶有高度同源性，两者均含有半胱氨酸蛋白酶活性位点处的保守性氨基酸序列, 因此cysA, cysB均为镰形扇头蜱两个新的组织蛋白酶L-样半胱氨酸蛋白酶基因。RT-PCR分析表明，CysA和CysB在镰形扇头蜱的不同发育阶段表达情况不一。

Molecular Cloning of Two Rhipicephalus haemaphysaloides haemaphysaloides Cathepsin L-like Cysteine Proteinase Gene

Ticks are obligate ectoparasites and vectors of arboviruses,vickettsiate, spiroc hetes and parasitil protozoa of humans and domestic animals. Immunological prote ction of mammalian hosts against tick infestation has been proposed as the most sustainable alternative tick control method to the current use of acaricides. Th e success of this method is dependent on the identification of key molecules for use as tick vaccine antigens. Proteolytic enzymes are involved in a wide range of cellular processes, thus they can be considered as good target antigens for a tick vaccine. In the present study, we used rapid amplification of cDNA ends pr otocol and primers that were designed based on the consensus amino acid motifs f lanking present in all papain-like cysteine proteinases, to amplify, sequence a nd characterize two Rhipicephalus haemaphysaloides haemaphysaloides cathepsi n L-like cysteine proteinases, named as cysA and cysB. The full length of cysA is 1168bp, encoding a 332 amino acid residue polypeptide with 36 33 kD predicted molecular mass; the full length of cysB is 1153bp, encoding a 335 a mino acid residue polypeptide with 37 56kD predicted molecular mass. The consen sus amino acid motifs flanking presence in both deduced amino acid sequences. An d both genes show high sequence homology to other tick cathepsin L-like cystein e proteinase, so they were identified as members of the cysteine proteinase gene family. Expression analysis by RT-PCR revealed that cysA and cysB were expressed differently in different periods of tick development.