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Abstract [Objectives] This study was conducted to investigate the chemical synthesis of peptide κ-CTx-btg02.
[Methods]Linear peptide κ-CTx-btg02 was synthesized by solid phase peptide synthesis （SPPS）. After oxidation and folding of the linear peptide， mass spectrometry identification and high performance liquid chromatography （HPLC） purification were performed. Then， the MTT method and insect injection method were applied to study its insecticidal activity.
[Results] The peptide κ-CTx-btg02 was successfully synthesized by the SPPS method， and identified for the formation of disulfide bonds by mass spectrometry identification， and the purity after HPLC separation was greater than 95%. The MTT experiment showed that the peptide κ-CTx-btg02 could inhibit the growth of insect cells sf9， with a half effective dose of 0.13 nM. The insect injection experiment showed that the peptide κ-CTx-btg02 could effectively kill Tenebrio molitor， with a half lethal dose of 15.6 nM. The results of the electrophysiological experiment showed that 10 μM peptide κ-CTx-btg02 had no blocking activity on murine acetylcholine α2β3 and α3β4. Therefore， the peptide κ-CTx-btg02 had a good inhibitory effect on the growth of insect cells， highly effective insecticidal activity and weak mammalian toxicity.
[Conclusions]This study lays a foundation for the development of new， safe and efficient peptide biological insecticides.
Key words Peptide; Solid phase peptide synthesis; Oxidative folding; Insecticidal activity; Electrophysiology
Received： May 3， 2021 Accepted： July 2021
Supported by Natural Science Foundation of Hainan Province （820RC636）.
Qin CHEN （1983-）， female， P. R. China， lecturer， devoted to research about marine resource development.
*Corresponding author. E-mail： [email protected].
In recent years， with the abuse of chemical pesticides， the problem of agricultural pollution has become more and more serious， which directly threatens the safety of agricultural ecology and the sustainable development of agriculture， which in turn brings food safety problems to human health[1]. How to solve the agricultural environmental crisis and ecological security issues is imminent， so the demand for new， efficient and safe biological pesticides is very urgent[2-3]. At present， animals that prey on insects can usually secrete peptide toxins， which can kill and capture prey through the ion channels or receptors of specific insects， but their toxicity to mammals is very small or has no toxic side effects[4-5]. It has been reported in literatures that cone snails， sea anemones， scorpions， spiders and predatory mites can secrete peptide toxins[6-9]. Therefore， the search for insecticidal active ingredients from marine or terrestrial animal peptide toxins is currently one of the hotpots in the research of safe pesticides. In this study， the linear peptide κ-CTx-btg02 was synthesized by solid phase peptide synthesis （SPPS）， and oxidized by the two-step oxidation method to oxidized folded peptide κ-CTx-btg0 which was then purified by high performance liquid chromatography for identification by mass spectrometry， and the insecticidal activity of the peptide was tested by the MTT method and insect injection method， laying a foundation for the development of new， efficient and safe biological pesticides. Electrophysiological activity
In this study， we tested the blocking activity of the peptide κ-CTx-btg02 on the 2 subtypes of murine acetylcholine receptors （nAChRs）. The results are shown in Fig. 5. It can be seen that 10 μmol/L peptide κ-CTx-btg01 had no effects on murine α3β4 and α2β3 nAChRs， revealing that the conotoxin κ-CTx-btg02 is less toxic to mammals.
Discussion
In recent years， the wanton abuse of chemical pesticides has led to environmental pollution by pesticide residues and pest resistance， and also poisoned the natural enemies of pests， thus destroying the ecological balance. Insect virus insecticides have the advantages of being safe against natural enemies of pests， being environmentally friendly and no production of drug resistance， so they are widely used[12]. However， insect virus insecticides have fatal disadvantages such as narrow insecticidal spectrum and slow insecticidal speed. With the development of molecular biology and genetic engineering， the use of exogenous genes to modify insect viruses has become an effective way to overcome its shortcomings[13-14]. Using insectivorous peptide genes as synergistic genes to construct recombinant baculoviruses can effectively solve the shortcomings of chemical pesticides and wild-type baculovirus insecticides， laying a foundation for the development of new， efficient and safe recombinant insect virus insecticides.
At present， it has been reported that peptide toxins of invertebrates such as cone snails， sea anemones， scorpions， spiders and predatory mites can bind to targets on insect nerve cell membranes and can specifically paralyze and poison insects[6-9]. Among them， previous studies have found that conotoxin can specifically act on insects and have insecticidal ability， and have little toxicity or no toxic side effects to mammals[4，6]. Conotoxin is mainly used in chronic pain， epilepsy and cancer. For example， MVIIA has been approved by the FDA as an analgesic for advanced cancer[15-18]. The peptide κ-CTx-btg022 is derived from the Hainan Conus betulinus. It has 2 disulfide bonds formed by 4 cysteines， and it is speculated through homology comparison that it can act on the acetylcholine receptor and has an insecticidal activity. In this study， the peptide κ-CTx-btg02 was synthesized by SPPS method and two-step oxidation folding method， and then the insecticidal activity was studied by MTT method， insect injection method and electrophysiology. The experimental results showed that the peptide κ-CTx-btg02 had the activity of inhibiting the growth of insect cells sf9， with a half effective dose of 0.13 nM; and it had insecticidal activity against T. molitor， with a half lethal dose of 15.6 nM. The electrophysiological results showed that the peptide κ-CTx-btg02 had no blocking effect on murine acetylcholine， suggesting that it has less toxicity to mammals， thus confirming that it has high safety characteristics for humans. Therefore， it was found from this study that the peptide κ-CTx-btg02 can specifically act on insects and has insecticidal ability， and has little toxicity or no toxic side effects to mammals. It will become the most potential insect baculovirus synergistic gene. References
[1] ROBINSON SA， RICHARDSON SD， DALTON RL， et al. Assessment of sublethal effects of neonicotinoid insecticides on the life-history traits of 2 frog species[J]. Environ Toxicol Chem， 2019（7）： 4511.
[2] HAASE S， SCIOCCO-CAP A， ROMANOWSKI V. Baculovirus insecticides in Latin America： historical overview， current status and future perspectives[J]. Viruses， 2015， 7（5）： 2230-2267.
[3] GORMAN K， HEWITT F， DENHOLM I， et al. New developments in insecticide resistance in the glasshouse whitefly （Trialeurodes vaporariorum） and the two-spotted spider mite （Tetranychus urticae） in the UK[J]. Pest Manag Sci， 200 58（2）： 123-130.
[4] GAO B， PENG C， LIN B， et al. Screening and validation of highly-efficient insecticidal conotoxins from a transcriptome-based dataset of Chinese tubular cone snail[J]. Toxins， 2017（9）： 214.
[5] GAO B， ZHANGSUN D， WU Y， et al. Expression， renaturation and biological activity of recombinant conotoxin GeXIVAWT[J]. Appl Microbiol Biotechnol， 2013， 97（3）： 1223-1230.
[6] WU XY， AN TT， GAO BM. Study on chemical synthesis and insecticidal activity of conotoxin ImI[J]. Natural Product Research and Development， 2018， 30（12）： 2203-2206.
[7] FU Y， LI X， DU J， et al. Regulation analysis of AcMNPV-mediated expression of a Chinese scorpion neurotoxin under the IE P10 and PH promoter in vivo and its use as a potential bio-insecticide[J]. Biotechnol Lett， 2015， 37（10）： 1929-1936.
[8] KING GF， HARDY MC. Spider-venom peptides： structure， pharmacology， and potential for control of insect pests[J]. Annu Rev Entomol， 2013（58）： 475-496.
[9] TOMALSKI MD， MILLER LK. Insect paralysis by baculovirus-mediated expression of a mite neurotoxin gene[J]. Nature， 199 352（6330）： 82-85.
[10] WU Y， XU P， REN J， et al. Study on oxidative folding pathway of α-conotoxin TxIB[J]. Chinese Journal of marine druds， 2016， 35（4）： 65-70.
[11] WU X， TAE HS， HUANG YH， et al. Stoichiometry dependent inhibition of rat α3β4 nicotinic acetylcholine receptor by the ribbon isomer of α-conotoxin AuIB[J]. Biochemical pharmacology， 2018（155）： 288-297.
[12] GARZO E， MORENO A， PLAZA M， et al. Feeding behavior and virus-transmission ability of insect vectors exposed to systemic insecticides[J]. Plants （Basel）， 2020， 9（7）： 895.
[13] GAO B， PENG C， LIN B， et al. Screening and validation of highly-efficient insecticidal conotoxins from a transcriptome-based dataset of Chinese tubular cone snail[J]. Toxins， 2017（9）： 214.
[14] KHAN SA， ZAFAR Y， BRIDDON RW， et al. Spider venom toxin protects plants from insect attack[J]. Transgen. Res， 2006（15）： 349-357.
[15] HUSSAr DA. New drugs： Tigecycline， ziconotide， and clofarabine[J]. J Am Pharm Assoc， 2005， 45（5）： 636-639.
[16] GAO B， ZHANGSUN D， HU Y， et al. Expression and secretion of functional recombinant μO-Conotoxin MrVIB-His-tag in Escherichia coli[J]. Toxicon， 2013（72）： 81-89.
[17] PRASHANTH JR， BRUST A， JIN AH， et al. Cone snail venomics： From novel biology to novel therapeutics[J]. Future Med Chem， 2014， 6（15）： 1659-1675.
[18] GAO B， PENG C， YANG J， et al. Cone snails： A big store of conotoxins for novel drug discovery[J]. Toxins， 2017（9）： 397.
[Methods]Linear peptide κ-CTx-btg02 was synthesized by solid phase peptide synthesis （SPPS）. After oxidation and folding of the linear peptide， mass spectrometry identification and high performance liquid chromatography （HPLC） purification were performed. Then， the MTT method and insect injection method were applied to study its insecticidal activity.
[Results] The peptide κ-CTx-btg02 was successfully synthesized by the SPPS method， and identified for the formation of disulfide bonds by mass spectrometry identification， and the purity after HPLC separation was greater than 95%. The MTT experiment showed that the peptide κ-CTx-btg02 could inhibit the growth of insect cells sf9， with a half effective dose of 0.13 nM. The insect injection experiment showed that the peptide κ-CTx-btg02 could effectively kill Tenebrio molitor， with a half lethal dose of 15.6 nM. The results of the electrophysiological experiment showed that 10 μM peptide κ-CTx-btg02 had no blocking activity on murine acetylcholine α2β3 and α3β4. Therefore， the peptide κ-CTx-btg02 had a good inhibitory effect on the growth of insect cells， highly effective insecticidal activity and weak mammalian toxicity.
[Conclusions]This study lays a foundation for the development of new， safe and efficient peptide biological insecticides.
Key words Peptide; Solid phase peptide synthesis; Oxidative folding; Insecticidal activity; Electrophysiology
Received： May 3， 2021 Accepted： July 2021
Supported by Natural Science Foundation of Hainan Province （820RC636）.
Qin CHEN （1983-）， female， P. R. China， lecturer， devoted to research about marine resource development.
*Corresponding author. E-mail： [email protected].
In recent years， with the abuse of chemical pesticides， the problem of agricultural pollution has become more and more serious， which directly threatens the safety of agricultural ecology and the sustainable development of agriculture， which in turn brings food safety problems to human health[1]. How to solve the agricultural environmental crisis and ecological security issues is imminent， so the demand for new， efficient and safe biological pesticides is very urgent[2-3]. At present， animals that prey on insects can usually secrete peptide toxins， which can kill and capture prey through the ion channels or receptors of specific insects， but their toxicity to mammals is very small or has no toxic side effects[4-5]. It has been reported in literatures that cone snails， sea anemones， scorpions， spiders and predatory mites can secrete peptide toxins[6-9]. Therefore， the search for insecticidal active ingredients from marine or terrestrial animal peptide toxins is currently one of the hotpots in the research of safe pesticides. In this study， the linear peptide κ-CTx-btg02 was synthesized by solid phase peptide synthesis （SPPS）， and oxidized by the two-step oxidation method to oxidized folded peptide κ-CTx-btg0 which was then purified by high performance liquid chromatography for identification by mass spectrometry， and the insecticidal activity of the peptide was tested by the MTT method and insect injection method， laying a foundation for the development of new， efficient and safe biological pesticides. Electrophysiological activity
In this study， we tested the blocking activity of the peptide κ-CTx-btg02 on the 2 subtypes of murine acetylcholine receptors （nAChRs）. The results are shown in Fig. 5. It can be seen that 10 μmol/L peptide κ-CTx-btg01 had no effects on murine α3β4 and α2β3 nAChRs， revealing that the conotoxin κ-CTx-btg02 is less toxic to mammals.
Discussion
In recent years， the wanton abuse of chemical pesticides has led to environmental pollution by pesticide residues and pest resistance， and also poisoned the natural enemies of pests， thus destroying the ecological balance. Insect virus insecticides have the advantages of being safe against natural enemies of pests， being environmentally friendly and no production of drug resistance， so they are widely used[12]. However， insect virus insecticides have fatal disadvantages such as narrow insecticidal spectrum and slow insecticidal speed. With the development of molecular biology and genetic engineering， the use of exogenous genes to modify insect viruses has become an effective way to overcome its shortcomings[13-14]. Using insectivorous peptide genes as synergistic genes to construct recombinant baculoviruses can effectively solve the shortcomings of chemical pesticides and wild-type baculovirus insecticides， laying a foundation for the development of new， efficient and safe recombinant insect virus insecticides.
At present， it has been reported that peptide toxins of invertebrates such as cone snails， sea anemones， scorpions， spiders and predatory mites can bind to targets on insect nerve cell membranes and can specifically paralyze and poison insects[6-9]. Among them， previous studies have found that conotoxin can specifically act on insects and have insecticidal ability， and have little toxicity or no toxic side effects to mammals[4，6]. Conotoxin is mainly used in chronic pain， epilepsy and cancer. For example， MVIIA has been approved by the FDA as an analgesic for advanced cancer[15-18]. The peptide κ-CTx-btg022 is derived from the Hainan Conus betulinus. It has 2 disulfide bonds formed by 4 cysteines， and it is speculated through homology comparison that it can act on the acetylcholine receptor and has an insecticidal activity. In this study， the peptide κ-CTx-btg02 was synthesized by SPPS method and two-step oxidation folding method， and then the insecticidal activity was studied by MTT method， insect injection method and electrophysiology. The experimental results showed that the peptide κ-CTx-btg02 had the activity of inhibiting the growth of insect cells sf9， with a half effective dose of 0.13 nM; and it had insecticidal activity against T. molitor， with a half lethal dose of 15.6 nM. The electrophysiological results showed that the peptide κ-CTx-btg02 had no blocking effect on murine acetylcholine， suggesting that it has less toxicity to mammals， thus confirming that it has high safety characteristics for humans. Therefore， it was found from this study that the peptide κ-CTx-btg02 can specifically act on insects and has insecticidal ability， and has little toxicity or no toxic side effects to mammals. It will become the most potential insect baculovirus synergistic gene. References
[1] ROBINSON SA， RICHARDSON SD， DALTON RL， et al. Assessment of sublethal effects of neonicotinoid insecticides on the life-history traits of 2 frog species[J]. Environ Toxicol Chem， 2019（7）： 4511.
[2] HAASE S， SCIOCCO-CAP A， ROMANOWSKI V. Baculovirus insecticides in Latin America： historical overview， current status and future perspectives[J]. Viruses， 2015， 7（5）： 2230-2267.
[3] GORMAN K， HEWITT F， DENHOLM I， et al. New developments in insecticide resistance in the glasshouse whitefly （Trialeurodes vaporariorum） and the two-spotted spider mite （Tetranychus urticae） in the UK[J]. Pest Manag Sci， 200 58（2）： 123-130.
[4] GAO B， PENG C， LIN B， et al. Screening and validation of highly-efficient insecticidal conotoxins from a transcriptome-based dataset of Chinese tubular cone snail[J]. Toxins， 2017（9）： 214.
[5] GAO B， ZHANGSUN D， WU Y， et al. Expression， renaturation and biological activity of recombinant conotoxin GeXIVAWT[J]. Appl Microbiol Biotechnol， 2013， 97（3）： 1223-1230.
[6] WU XY， AN TT， GAO BM. Study on chemical synthesis and insecticidal activity of conotoxin ImI[J]. Natural Product Research and Development， 2018， 30（12）： 2203-2206.
[7] FU Y， LI X， DU J， et al. Regulation analysis of AcMNPV-mediated expression of a Chinese scorpion neurotoxin under the IE P10 and PH promoter in vivo and its use as a potential bio-insecticide[J]. Biotechnol Lett， 2015， 37（10）： 1929-1936.
[8] KING GF， HARDY MC. Spider-venom peptides： structure， pharmacology， and potential for control of insect pests[J]. Annu Rev Entomol， 2013（58）： 475-496.
[9] TOMALSKI MD， MILLER LK. Insect paralysis by baculovirus-mediated expression of a mite neurotoxin gene[J]. Nature， 199 352（6330）： 82-85.
[10] WU Y， XU P， REN J， et al. Study on oxidative folding pathway of α-conotoxin TxIB[J]. Chinese Journal of marine druds， 2016， 35（4）： 65-70.
[11] WU X， TAE HS， HUANG YH， et al. Stoichiometry dependent inhibition of rat α3β4 nicotinic acetylcholine receptor by the ribbon isomer of α-conotoxin AuIB[J]. Biochemical pharmacology， 2018（155）： 288-297.
[12] GARZO E， MORENO A， PLAZA M， et al. Feeding behavior and virus-transmission ability of insect vectors exposed to systemic insecticides[J]. Plants （Basel）， 2020， 9（7）： 895.
[13] GAO B， PENG C， LIN B， et al. Screening and validation of highly-efficient insecticidal conotoxins from a transcriptome-based dataset of Chinese tubular cone snail[J]. Toxins， 2017（9）： 214.
[14] KHAN SA， ZAFAR Y， BRIDDON RW， et al. Spider venom toxin protects plants from insect attack[J]. Transgen. Res， 2006（15）： 349-357.
[15] HUSSAr DA. New drugs： Tigecycline， ziconotide， and clofarabine[J]. J Am Pharm Assoc， 2005， 45（5）： 636-639.
[16] GAO B， ZHANGSUN D， HU Y， et al. Expression and secretion of functional recombinant μO-Conotoxin MrVIB-His-tag in Escherichia coli[J]. Toxicon， 2013（72）： 81-89.
[17] PRASHANTH JR， BRUST A， JIN AH， et al. Cone snail venomics： From novel biology to novel therapeutics[J]. Future Med Chem， 2014， 6（15）： 1659-1675.
[18] GAO B， PENG C， YANG J， et al. Cone snails： A big store of conotoxins for novel drug discovery[J]. Toxins， 2017（9）： 397.