Cloning and expression of Ripicephalus (Boophilus) annulatus cathepsin L Sattari Tabrizi.S, Nabian.S, Ebrahimzadeh .E, Shayan.P, Amininia.N
عنوان دوره: دومین کنگره بین المللی حشره شناسی ایران
نویسندگان
چکیده
Introduction:
Ticks are important ectoparasites which can cause massive losses in livestock worldwide due to direct damages such as blood loss and pathogens transmission. The tick Rhipicephalus (Boophilus) annulatus is one of the most important bovine ectoparasites, which can transmit the protozaoans Babesia Spp, and Anaplasma Spp to cattle.Cysteine proteases are involved in several host-tick interactions including invasion of host tissues, immune evasion, pathogens transmission, embryogenesis and blood digestion. Cathepsin as a cysteine protease could be used like a target for chemotherapy or an immunogen in vaccine production. In this study, the gene encoding of R. annulatus cathepsin L-like enzyme (RaCL1) was cloned into PQ30 vector and expressed in E.coli Bl21
Materials and methods:
The Fully engorged female of R.annulatus tick were collected from cattle’s in north of Iran
These ticks were incubated in 280 C and 85% humidity for almost 45 days until hatching and the larvae were stored in -700C for further uses. cDNA was synthesized from isolated RNA of larva and RT-PCR product was cloned in pTZ57R/T Vector. The coding region was excised by digestion with Hind III and Bam HI in both RT-Pcr product and PQE-30 plasmid. RT-Pcr product was ligated in to PQE-30 and The DNA fragment was transferred and expressed in E.coli Bl21.
Results:
The encoding gene of R. annulatus cathepsin L-like enzyme (RaCL1) was cloned into PQE-30 vector. Sequencing showed the nucleotide length of 999 bp. The DNA fragment corresponding the encoding gene was transferred to the E.coli BL21 and expressed with 0.2 mM IPTG at 37 0 c for 4 hours. Bioinformatics analysis showed 332 amino acids with an approximate molecular weight of 36.3 kDa for resulting protein in SDS-PAGE analysis.
Discussion:
The most common method for prevention and control of tick infestation is using acaricide. Destructive environmental impact caused many efforts take placed to utilize other policy, like vaccination. Now a day there are two commercial vaccines against cattle’s ticks: TickGARD Plus® in Australia and Gavac® in Cuba. However, they are not completely effective in other tick’s strains in the world. Based on importance role of cysteine protease like cathepsine in tick’s life cycle e.g. development and hemoglobin digestion, characterization and studying their role in biological procedures are crucial for control strategies. In this study, the authors tried to clone and expressed the cathepsin gene of R. annulatus and hope to find a way to control tick’s infestation in the future.
Ticks are important ectoparasites which can cause massive losses in livestock worldwide due to direct damages such as blood loss and pathogens transmission. The tick Rhipicephalus (Boophilus) annulatus is one of the most important bovine ectoparasites, which can transmit the protozaoans Babesia Spp, and Anaplasma Spp to cattle.Cysteine proteases are involved in several host-tick interactions including invasion of host tissues, immune evasion, pathogens transmission, embryogenesis and blood digestion. Cathepsin as a cysteine protease could be used like a target for chemotherapy or an immunogen in vaccine production. In this study, the gene encoding of R. annulatus cathepsin L-like enzyme (RaCL1) was cloned into PQ30 vector and expressed in E.coli Bl21
Materials and methods:
The Fully engorged female of R.annulatus tick were collected from cattle’s in north of Iran
These ticks were incubated in 280 C and 85% humidity for almost 45 days until hatching and the larvae were stored in -700C for further uses. cDNA was synthesized from isolated RNA of larva and RT-PCR product was cloned in pTZ57R/T Vector. The coding region was excised by digestion with Hind III and Bam HI in both RT-Pcr product and PQE-30 plasmid. RT-Pcr product was ligated in to PQE-30 and The DNA fragment was transferred and expressed in E.coli Bl21.
Results:
The encoding gene of R. annulatus cathepsin L-like enzyme (RaCL1) was cloned into PQE-30 vector. Sequencing showed the nucleotide length of 999 bp. The DNA fragment corresponding the encoding gene was transferred to the E.coli BL21 and expressed with 0.2 mM IPTG at 37 0 c for 4 hours. Bioinformatics analysis showed 332 amino acids with an approximate molecular weight of 36.3 kDa for resulting protein in SDS-PAGE analysis.
Discussion:
The most common method for prevention and control of tick infestation is using acaricide. Destructive environmental impact caused many efforts take placed to utilize other policy, like vaccination. Now a day there are two commercial vaccines against cattle’s ticks: TickGARD Plus® in Australia and Gavac® in Cuba. However, they are not completely effective in other tick’s strains in the world. Based on importance role of cysteine protease like cathepsine in tick’s life cycle e.g. development and hemoglobin digestion, characterization and studying their role in biological procedures are crucial for control strategies. In this study, the authors tried to clone and expressed the cathepsin gene of R. annulatus and hope to find a way to control tick’s infestation in the future.
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