| 其他摘要 | Ticks are obligate haematophagous ectoparasites and second only to mosquitoes as vectors of pathogens causing human and animal disease. They are capable of transmitting a variety of pathogens involved in some infectious diseases, such as human lyme disease, babesiosis, spotted fever and so on. The salivary glands of ticks can secrete a diversity of active components to resist the host immune response and can also release toxins, result in paralysis of the infested host. At present, there are few reports about the neurotoxins from salivary glands of hard ticks. In this thesis, a series of experiments were carried out to study the structure and function of neurotoxin ISTX-I from Ixodes scapularis. A neurotoxin ISTX-I from Ixodes scapularis was successfully expressed by the pET expression system in E.coli. After Ni-chelating affinity chromatography, formic acid cutting, Sephadex G-50, AKTA Resource Q anion exchange chromatography and RP-HPLC, we got purified ISTX-I polypeptide. By MALDI-TOF-MS detection, its molecular weight is 4825.3 Da, consistent with the theoretical molecular weight. The neurotoxin ISTX-I has six cysteines, which have formed three pairs of disulfide bond. The disulfide linkage of ISTX-I was determined between Cys3-Cys15, Cys9-Cys29 and Cys14-Cys39, adopting a 1-4, 2-5 and 3-6 disulfide bond pattern, by means of partial reduction, Edman degradation sequencing and trypsin digestion. The effects of ISTX-I on voltage-gated K+, Ca2+ and Na+ channels were investigated by whole-cell patch clamp technique. The results revealed that ISTX-I had no effect on voltage-gated potassium channels, calcium channels and TTX-R sodium channels of dorsal root ganglion. However it can obviously inhibit TTX-S sodium channels of DRG neurons and voltage-gated sodium channels Nav1.7, the IC50 value was 2.47 ± 1.47 μM. The further detection of its activity on the subtypes of voltage-gated sodium channels (Nav1.1-Nav1.7), found that ISTX-I selectively inhibit Nav1.7 (IC50 = 1.60 ± 0.18 μM), but it had no significant effect on Nav1.1, Nav1.2, Nav1.3, Nav1.4, Nav1.5 and Nav1.6. The analysis of its effects on the activation and inactivation kinetics of TTX-S sodium channels and voltage-gated sodium channels Nav1.7 showed that ISTX-I could not affect their activation curves, but it shifted their inactivation curves. The inactivation curves of TTX-S sodium channels and voltage-gated sodium channels Nav1.7 were shifted for about 11.22 mV and 6.11 mV in hyperpolarizing direction, respectively. This reduced the threshold of inactivation of the channels and shortened the opening time of the channels. This paper found a voltage-gated sodium channels Nav1.7 inhibitor (ISTX-I) from the salivary glands of Ixodes scapularis for the first time and its disulfide bond pattern was identified. The results will provide new ideas for the research of tick toxins and a certain theoretical basis as to explaining the forming mechanism of tick paralysis. |
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