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Molecular Cloning And Analysis Of Ferritin And Antimicrobial Peptide CDNA Of Ticks

Posted on:2005-01-26Degree:MasterType:Thesis
Country:ChinaCandidate:X BaiFull Text:PDF
GTID:2120360122994756Subject:Basic veterinary
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1.Molecular cloning and analysis of ferritin of Boophilusmicroplus:A gene encoding ferritin was isolated and cloned from Boophilus microplus. The cDNA is composed of 642 nucleotides and encoding 172 amino-acid residues, the encoding region is between 123 to 639 nt, the deduced mass is 19.9kDa and isoelectric point is 4.24.The deduced amino-acid sequence shows 93.60%, 88.37% and 83.72% homology with the ferritin from tick Dermacentor variabilis, Ornithodoros moubata and Ixoded ricinus respectively. The stem-loop structure of a putative iron responsive element is found in the 5'-untranslated region of ferritin mRNA. The protein contains the conserved motifs for ferroxidase center. RT-PCR analysis suggests the ferritins are expressed in the eggs, larvas. fed female adult, engorgement female adult and male adult ticks of B. microplus. The encoding region was subcloned into vector pET- 28a(+),and expressed in E.coli BL21 (DE3) .The recombinant protein shows a single band by SDS-PAGE, the size is about 25kDa. Western blot analysis suggests the rabbit anti- B.microplus salivary serum can recognize recombinant protein.2,Molecular cloning and expression of ferritin of Rhipicephalushaemaphysaloides haemaphysaloides:A gene encoding ferritin was isolated and cloned from Rhipicephalus haemaphysaloides haemaphysaloides. The cDNA is composed of 642 nucleotides and encoding 172 ammo-acid residues, the encoding region is between 123 to 639 nt, the deduced mass is 19.9kDa and isoelectric point is 4.90.The deduced amino-acid sequence shows 98.26% , 97.09%, 96.51 % and95.93% homology with the ferritin from tick Hyalomma asiaticum asiatium, Dermacentor variabilis, Amblyomma americanum and Boophilus microplus respectively. The stem-loop structure of a putative iron responsive element is found in the 5'-untranslated region of ferritin mRNA. The protein contains the conserved motifs for ferroxidase center. RT-PCR analysis suggests the ferritins are expressed in the eggs, larvae, engorgement larvae, engorgement nymphae, adult ticks, tick salivary gland, tick shells of R. haemaphysaloides haemaphysaloides. The encoding region was subcloned into vector pET-28a(+), and expressed in E.coli BL21 (DE3) .The recombinant protein shows a single band by SDS-PAGE, the size is about 25kDa.3,Molecular cloning and analysis of antimicrobial peptide ofBoophilus microplus:To study the mechanism between ticks and pathogeny and control tick and tick-borne diseases, On the basis of the lately nucleotide sequence of structural specially microplusin from Brazil B. microplus, a gene encoding antimicrobial peptide was isolated and cloned from B. microplus of Anhui, China. It is composed of 383 nucleotides and encoding 110 amino-acid residues, the deduced mass is 12.2 kDa, isoelectric point is 4.87. The sequence has 100 % homology from the microplusin of Brazil B. microplus. RT-PCR analysis suggests the antimicrobial peptide are expressed in the eggs, larvas, fed female adult, engorgement female adult and male adult ticks of B. microplus. After cleaving the signal peptide the residual was subcloned into expressing vector pET-28a(+), and expressed in E.coli BL21 (DE3 ) . The recombinant protein shows a single band by SDS-PAGE, the size is about 15 kDa. Antimicrobial experimentation in vitro shows that it seems no activity. Western blotting analysis suggests the rabbit anti-B.microplus salivary serum can recognize recombinant protein.
Keywords/Search Tags:Boophilus microplus, Rhipicephalus haemaphysaloides haemaphysaloides, ferrtin, antimicrobial peptide, cDNA cloning, recombinant expression
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