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Argiope spider toxin extraction and preparation of crude


Of: Chen with water, Zhang Hao, YUAN Feng-hui, Mao Yuexiang, Cao Ning, Zhu Mang
Abstract aims to master the habit and bright gold spider morphological characteristics of gland morphology and body mass correlation optimize specific manufacturing line, get a higher biological activity than the pure spider crude drug. Methods venom morphological and anatomical, with the help of mathematical statistics to analyze the venom gland morphology and spider body mass correlation; using venom gland dissection with direct access to the nature of stable and up to the amount of spider toxin crude to chicken red blood cells as a model test the venom potency, Explore the toxins inhibit the type of ion channels. Results gland weight and body mass were positively correlated, in accordance with the optimized preparation and technical lines, the system was 5 mg Argiope spider venom; Argiope spider toxin to promote erythrocyte agglutination and cracking role in the calcium ion environment toxin on red blood cells Cracking stronger. Conclusion gland dissection can be a lot higher purity of spiders the most poisonous, poisonous spider which initially identified the lectin and phospholipase containing two ingredients.
Key words Argiope amoena; gland anatomy; venom; cell toxicity
ChinaAbstract: ObjectiveHaving a detailed mastery about the Argiope amoena 's morphological characteristics and living habits, we analyzed the relevence of poison gland weight and length to its body. Using the optimized method we obtained higher biological properties and more pure venom . Methods We got the relevence of poison gland weight and length to its body using statistical method by anatoming poison gland. Isolating poison gland from spider, obtained stable and maximum amount of venom, we tested titer of venom and explore the type of inhibit ion channels toxin using chicken red blood cells as a model. Results Venom gland 's weight and body mass were positively correlated. With the optimized mean prepared method, five millingrams of crude venom were got. The venom has effect of promoting red blood cell aggregating and cracking. The blood cell cracked more seriously in the Calcium ions environment. Conclusion More qualified venom can be got by venom gland dissection. We initially know that venom contains phospholipase lectin and agglutinin.
Key words: Argiope amoena; Anatomy of venom; Crude toxin; Cell physiology
Spider is an important pest natural enemies, their toxins to kill their prey paralysis. Spider toxin complex components, mainly proteins and multi-amine, protein, including peptide neurotoxins, cytotoxins, enzymes, kinin analogs, peptide and peptide aggregation, and many more amines are ATP, the nucleotide , amino acids, polyamines, amine and other low molecular weight substances and inorganic salts. Spider toxin with a variety of activities, has been found to contain a variety of specific peptide ligand, can selectively and K +,[link widoczny dla zalogowanych], Na +, Ca2 + channel subtypes combined [1 ~ 11]. Liang Song domestic equality [12] Huwentoxin Hainan and Guangxi Tarantula spider toxins were more in-depth research. Argiope spider Argiope amoena under Araneae genus Argiope garden spiders, a large body, for the spider webs in the southern provinces of China were distributed, multi-infested in the summer and autumn to winter egg sac [13]. Because Argiope spider live in the lush vegetation in the mountains, not easy to keep, so on Argiope spider toxins rarely reported.
1 equipment and materials
1.1 devices
Shanghai Precision A1004 electronic balance, Olympus CX41 microscope camera system, Shanghai Xinmiao DNP-9052BS-Ⅲ electric heated incubator and DHG-9073BS-Ⅲ constant temperature drying oven, Hunan Star TGL-16B High-speed desktop centrifuge machine, Kunshan KQ-50DB CNC ultrasonic cleaning, Beijing Tech 0610-403 Stereo Microscope, Mitutoyo 560-108 NK20 caliper, Shanghai optical microscope and the micrometer Yahya Wing vacuum pump and dryer and so on.
1.2 Argiope spider collection
Argiope amoena Guixi City, Jiangxi Province,[link widoczny dla zalogowanych], collected from the cold forest, a total of 28, in which male 3, female 25. Argiope amoena is mainly distributed in the mountain foothills, on the sunny side rotary screen cloth to larger insect prey, hi networking on human habitation around the bush, especially cow pen near as much. Argiope amoena fragile appendage long, easily injured, in the acquisition process should take extra care; can catch insect net to catch the spider, into box, put some leaves in order to maintain humidity and good ventilation to avoid oscillation and excessive heat. After the acquisition is completed return should be implemented on the spider in a timely manner on the semi-shrub ecological stocking.
2 results and methods
2.1 Argiope spider venom gland morphology and body mass correlation analysis
Argiope spider's venom gland is located in the smallest chelate-based front end to the head extended, multi-anterior chest in the first period, covering the ganglion, on about 22 and tend to spider body relative longitudinal axis. For fast, accurate and efficient access to the spider venom to avoid material losses, we had a bright golden spider venom gland morphology anatomy, specifically the structure of venom and specific location, then venom for morphological description. Before dissection using electronic balance out the spider's weight, using a vernier caliper on the large individual difference measure out of its head and thorax and abdomen length and width, while the individual is small spiders with micrometer measure out of its head and thorax and abdomen The length and width, then measure the length and width venom gland removed, and weighed. Specific measurement results shown in Table 1 and 2, regression analysis of data shows that: Argiope spider in the same sex, same habitat, body size, not at the same time, the location and morphology of the spider venom and spider species evolution, lifestyle and patterns volume has a certain relationship, which, into the spider's venom gland weight and their body mass were positively correlated, significantly correlated; into the body of the gland length with the first thoracic length was also positively related to the significance test is very significant. Table 1 Argiope spider venom gland of adult weight and relevance of individual quality (slightly) Table 2 Argiope spider venom into the body length and individual length of data dependence analysis (omitted)
2.2 Argiope amoena Preparation of crude drug extract and optimization
As Argiope spider with large Huwentoxin, spiders and other King Charles Ying Mao compared to the adult is relatively small,[link widoczny dla zalogowanych], toxic small amount, and the limb long it difficult to adopt poison; we try to live induction to take drugs, electrical stimulation to take poison and venom gland and other anatomical separation method, determined by gland dissection,[link widoczny dla zalogowanych], after the comparison test after repeated, optimizing the amount of a drug is not suitable for anatomical uniform spider venom plasma drug laws take the steps below: first spider -4 ℃ refrigerator cold, dizziness, limb determined according to Ao gland tube position, and then from the back of the \carapace, by venom gland tube upward direction to find and carefully remove the tweezers, to lyophilization.
Take 15 on the dried venom, in 2 to 3 times the homogenate made in deionized water with 2 000 r / min rate of centrifuge 15 min, the supernatant. Canada, the sediment volume of deionized water, then centrifuged and the supernatant. Further adding an equal volume of sediments in deionized water, adjust pH value to 4.0, homogenized, centrifuged, and the supernatant combined supernatant, filtered to remove impurities, get mix with, to freeze, freezes, etc. block, the frozen ice spider poison placed in a vacuum dryer, vacuum drying, the bottom of the dryer put the right amount of calcium oxide as a desiccant, and then use the vacuum pump, pumping process,[link widoczny dla zalogowanych], to observe that if poisonous spider found in the surface of large bubbles, stop a moment apply again and again, until the basic dry particles into crystal sizes until 6 h of vacuum drying is generally completed within a complete drying. The spider toxin Serve dry powder or block, scraped into a vial of dry Dufen-packing, melting wax seal, label, indicating the spider toxin Dufen date and weight of the preparation, the package opaque black paper, put -5 ℃ refrigerator can save 1 year degeneration. Collecting venom and preparation of the whole process, should first pay attention to the whole process must be clean and sanitary, to ensure the purity of drug taken, followed by the process of collecting venom as far as possible at low temperature, all reagents must be pre-ice bath.
2.3 Argiope spider venom composition and potency testing
2.3.1 Argiope spider venom composition and structure of observation
Room temperature, extract preparation to the Argiope spider venom form of powder; using optical microscopy venom, 100 and 400 times as toxic under the thick sheet or sheet was uniform crystals, free of other particulate impurities. Figure 1.
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