Screening and Molecular Cloning of a Protective Antigen from the Midgut of Haemaphysalis longicornis

Abstract

Vaccination is considered a promising alternative for controlling tick infestations. Haemaphysalis longicornis midgut proteins separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membrane were screened for protective value against bites. The western blot demonstrated the immunogenicity of 92 kDa protein (P92). The analysis of the P92 amino acid sequence by LC-MS/MS indicated that it was a H. longicornis paramyosin (Hl-Pmy). The full lenghth cDNA of Hl-Pmy was obtained by rapid amplification of cDNA ends (RACE) which consisted of 2,783 bp with a 161 bp 3' untranslated region. Sequence alignment of tick paramyosin (Pmy) showed that Hl-Pmy shared a high level of conservation among ticks. Comparison with the protective epitope sequence of other invertebrate Pmy, it was calculated that the protective epitope of Hl-Pmy was a peptide (LEEAEGSSETVVEMNKKRDTE) named LEE, which was close to the N-terminal of Hl-Pmy protein. The secondary structure analysis suggested that LEE had non-helical segments within an α-helical structure. These results provide the basis for developing a vaccine against biting H. longicornis ticks.

Screening and Identification of Antigenic Proteins from the Hard Tick <i>Dermacentor silvarum</i> (Acari: Ixodidae)

“…Moreover, immunization of female BALB/c mice with recombinant protein NP-M1-HSP60 induced immune responses, and protected mice from lethal influenza H7N9 virus challenge and significantly inhibited viral replication [ 22 ]. In our previous studies [ 7 ], 66 kDa proteins from H. longicornis had immunogenic properties, which might be Hsp60 verified later by LC-MS/MS. We also found similar results in the experiments of screening antigenic proteins from D. silvarum .…”

“…The results of tick infestation challenge showed that rHlFER2 could significantly reduce the body weight of ticks infested from the rHlFER2-immunized rabbit compared to those from the control rabbits. Through mass spectrometry, Kim et al [ 6 ] and Hu et al [ 7 ] identified calreticulin and paramyosin (Pmy) from H. longicornis , respectively. These scholars have been trying to screen more effective antigenic proteins in the preparation of anti-tick vaccines.…”

“…Based on the molecular weight marker, the target protein bands were cut and placed into 2 ml tubes, respectively, for LC-MS/MS analysis. The method was the same as described previously [ 7 ].…”

“…By western blot analysis, 5 midgut membrane antigens with molecular weight 95, 85, 66, 49, and 42 kDa had been identified in Hyalomma anatolicum anatolicum [ 11 ]. Through mass spectrometry, calreticulin and Pmy were identified in H. longicornis [ 6 , 7 ]. By the similar method, 3 proteins (100, 68, and 52 kDa) from D. silvarum were demonstrated to have immunogenicity, and the results of LC-MS/MS showed that they might be Vg, Hsp60, and FBA ( Fig.…”

Paramyosin of canine Onchocerca lupi: usefulness for the diagnosis of a neglected zoonotic disease

“…By virtue of its major antigenic properties, this protein has been used for the development of diagnostic assays for filarioids of medical concern such as Wuchereria bancrofti and Brugia malayi [ 27 , 32 , 33 ]. Many studies of the paramyosin protein, as a potential vaccine candidate, have focussed on platyhelminths [ 34 – 39 ], with fewer studies on nematodes [ 40 , 41 ] and ticks [ 42 ]. An immunomodulatory effect of this protein has also been identified, inhibiting components of the complement cascade [ 43 , 44 ], binding of immunoglobulins and collagen [ 45 , 46 ].…”