Bovine immunoprotection against Rhipicephalus (Boophilus) microplus with recombinant Bm86-Campo Grande antigen / Imunoproteção de bovinos contra Rhipicephalus (Boophilus) microplus com antígeno recombinante Bm86-Campo Grande

The southern cattle fever tick, Rhipicephalus (Boophilus) microplus, is no doubt the most economically important ectoparasite of cattle globally. The inappropriate use of chemical acaricides has driven the evolution of resistance in populations of R. (B.) microplus. Anti-tick vaccines represent a technology that can be combined with acaricides in integrated control programs to mitigate the impact of R. (B.) microplus. The recombinant form of Bm86 antigen from the Campo Grande (rBm86-CG) strain of R. (B.) microplus was produced using the Pichiapastoris expression system to test its ability to immunoprotect cattle against tick infestation. Secretion of rBm86-CG by P. pastoris through the bioprocess reported here simplified purification of the antigen. A specific humoral immune response was detected by ELISA in vaccinated cattle. Immunoblot results revealed that polyclonal antibodies from vaccinated cattle recognized a protein in larval extracts with a molecular weight corresponding to Bm86. The rBm86-CG antigen showed 31% efficacy against the Campo Grande strain of R. (B.) microplus infesting vaccinated cattle. The rBm86-CG is an antigen that could be used in a polyvalent vaccine as part of an integrated program for the control of R. (B.) microplus in the region that includes Mato Grosso do Sul.

O carrapato Rhipicephalus (Boophilus) microplus é, sem dúvidas, o ectoparasito economicamente mais importante para o gado a nível mundial. A utilização inadequada de acaricidas tem impulsionado a evolução da resistência em populações de R. (B.) microplus. Vacinas contra o carrapato representam uma tecnologia que pode ser combinada com acaricidas em programas de controle integrado para diminuir o impacto de R. (B.) microplus. A forma recombinante da Bm86 da cepa Campo Grande (rBm86-CG) de R. (B.) microplus foi produzido utilizando o sistema de expressão em Pichia pastoris para testar sua capacidade de imunoproteção ao gado contra a infestação de carrapatos. A secreção de rBm86-CG em P. pastoris pelo bioprocesso, simplificou a purificação do antígeno. A resposta imune humoral específica foi detectada por ELISA em soros de bovinos vacinados. Resultados de "imunoblot" revelaram que anticorpos policlonais de bovinos vacinados reconheceram uma proteína em extratos de larvas com um peso molecular correspondente à Bm86. O antígeno rBm86-CG mostrou eficácia de 31% contra a amostra CG de R. (B.) microplus utilizada para infestar os bovinos vacinados. Pelos resultados obtidos, concluímos que a rBm86-CG é um antígeno que pode ser usado em uma vacina polivalente, como parte de um programa integrado para o controle de R. (B.) microplus no estado do Mato Grosso do Sul, Brasil.

Comparison of predicted binders in Rhipicephalus (Boophilus) microplus intestine protein variants BM86 Campo Grande strain, BM86 and BM95 / Comparação da previsão de ligação das variantes de proteína de intestino de Rhipicephalus (Boophilus) microplus Bm86 cepa Campo Grande, Bm86 e Bm95

This paper reports the sequence analysis of Bm86 Campo Grande strain comparing it with Bm86 and Bm95 antigens from the preparations TickGardPLUS and GavacTM, respectively. The PCR product was cloned into pMOSBlue and sequenced. The secondary structure prediction tool PSIPRED was used to calculate alpha helices and beta strand contents of the predicted polypeptide. The hydrophobicity profile was calculated using the algorithms from the Hopp and Woods method, in addition to identification of potential MHC class-I binding regions in the antigens. Pair-wise alignment revealed that the similarity between Bm86 Campo Grande strain and Bm86 is 0.2 percent higher than that between Bm86 Campo Grande strain and Bm95 antigens. The identities were 96.5 percent and 96.3 percent respectively. Major suggestive differences in hydrophobicity were predicted among the sequences in two specific regions.

O objetivo deste estudo foi analisar a seqüência de Bm86 cepa Campo Grande comparando-a com os antígenos Bm86 e Bm95 das preparações TickGardPLUS e GavacTM, respectivamente. O produto de PCR foi clonado em PMOSBlue e seqüenciado. Para calcular os conteúdos de alfa-hélice e fita beta do polipeptídio previsto, foi utilizada a ferramenta de prognóstico de estrutura secundária PSIPRED. O perfil de hidrofobicidade foi calculado usando os algoritmos de Hopp e Woods, além da identificação das possíveis regiões de ligação com MHC classe I nos antígenos. O alinhamento "pair-wise" revelou que a similaridade entre Bm86 cepa Campo Grande e Bm86 é 0,2 por cento maior que aquela entre Bm86 cepa Campo Grande e Bm95. As identidades foram de 96,5 por cento e 96,3 por cento, respectivamente. Com relação à hidrofobicidade, os resultados sugerem que a maior diferença entre as seqüências está localizada em duas regiões específicas.

Prokaryotic Expression of Bm86 Gene of Boophilus microplus and Optimization of the Expression Condition / 中国寄生虫学与寄生虫病杂志

A pair of specific primers was designed based on the reported Bm86 gene of Boophilus microplus,the Bm86 gene was cloned by PCR using the plasmid pMD18-T-Bm86 as templates,and subcloned into the prokaryotic plasmid pGEX-4T-1.The recombined plasmid was transformed into E.coli BL21(DE3) and followed by expression of the protein induced by different concentration of IPTG for different time.SDS-PAGE showed that the recombinant plasmid pGEX-4T-1/Bm86 expressed a fusion protein Bm86-GST(Mr 94 000) after being induced with IPTG.High level expre-ssion of Bm86-GST was found at 1 mmol/L IPTG condition after incubation for 8 h at 37 ℃,and the expression level of the recom-binant Bm86-GST reached up to 29% of total E.coli proteins.Western-blotting analysis showed that the recombinant Bm86-GST was recognized by the rabbit anti-B.microplus positive serum.