Crabs immune reinforcing agent high flux screening method
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A technology of immune enhancer and screening method, which is applied in the field of in vitro rapid screening of crab immune enhancers
Inactive Publication Date: 2010-02-10
ZHEJIANG INST OF FRESH WATER FISHERIES
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In previous studies, phenoloxidase activity and several other serum enzyme indicators were used as important indicators for the screening of crustacean immune enhancers, and were used to guide the evaluation of the enhancement effect of immune enhancers on animals. Literature reports on in vitro screening of activator as immune potentiator in crabs
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Embodiment 1
[0037] Example 1 Preparation of crab phenoloxidase crude extract:
[0038]1) Extract 2 times the volume of pre-cooled anticoagulant into a sterile syringe, use 75% alcohol cotton balls to sterilize the base of crab legs, and aseptically extract hemolymph from the base of crab legs;
[0039] 2) After centrifugation at 3000r / min at 4°C, resuspend the blood cells with 4°C pre-cooled sterile sodium cacodylate buffer (CAC buffer), centrifuge again, and add 0.1M pH 7.0 with 1 / 10 volume of hemolymph Resuspend the pellet in phosphate buffer, resuspend the pellet, and combine the cells in each tube;
[0040] 3) In an ice bath, use an ultrasonic cell disruptor to crush blood cells for 3-5 minutes. The specific conditions for crushing are: 200-250W power, oscillating for 30s, and intermittently for 15s. Continue like this for 3-5min until the cell suspension is basically transparent. 4°C 10000r / min Centrifuge for 20 minutes to obtain blood cell lysate (HLS), which is used as the crude e...
Embodiment 2
[0041] Example 2 Determination of Phenoloxidase Activity
[0042] ① Add 105 μL of 0.1M pH7.0 phosphate buffer (PB) to the 96-well plate, and 15 μL of the immunopotentiator to be tested;
[0043] ② Add 15 μL of the prepared HLS, mix well, and incubate at 25°C for 15 minutes;
[0044] ③ Add 15 μL of L-DOPA prepared with pH 7.0 PB, and immediately read the optical density value at 490 nm (OD 490nm );
[0045] ④Incubate the 96-well plate at 25°C and read the OD with a microplate reader every 5 minutes 490nm , measured for 15 minutes, and calculated the phenol oxidase activity unit (U).
[0046] 5. Calculate the phenoloxidase activity of each reaction well (treatment group), the calculation formula is:
[0047] u PO =(OD 0m -OD Tm ) / (Tm×0.001)
[0048] As the reaction time prolongs, the enzymatic reaction can gradually slow down, and the 96-well plate system takes a long time to add samples, which may cause errors in the initial reaction. It is recommended to use a reaction...
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Abstract
The invention relates to an extracorporeal fast screening method of the crabs immune reinforcing agent, in particular to a crabs immune reinforcing agent high flux screening method, comprising the following steps: mixing the immune reinforcing agent to be tested and crabs phenol oxidase original crude extract; activating the activity of the prophenoloxidase in the crabs original crude extract; converting the prophenoloxidase into active phenol oxidase; changing the color of the phenol oxidase catalyzing substrate L-dihydroxyphenylalanine from achromatic color into a color product-chinone; according to the coloration degree of the chinone, computing the activity enhancement rate of the immune reinforcing agent to be tested to the phenol oxidase to be taken as evaluation basis of the extracorporeal screening crabs immune reinforcing agent. By testing the activity of the phenol oxides with a microplate reader, the method automates the activity test of the phenol oxidase, greatly improvesthe screening efficiency of the crabs immune reinforcing agent, and realizes the high flux screening of the immune reinforcing agent. The method can provide fast and effective route for the extracorporeal screening of the crabs immune reinforcing agent, and is applicable to massive screening and prescreening of the activity of the crabs immune reinforcing agent.
Description
technical field [0001] The invention relates to an in vitro rapid screening method for crab immune enhancers. Background technique [0002] Seawater economic crabs are an important part of marine culture in my country. The main species of seawater crabs cultured in my country are Scylla serrata Forskal and Portunus sp, which belong to the order Crustacea and the order Decapoda. , Swimming crab family (Portunidae), widely distributed in the eastern coastal provinces of my country and Japan, India, Southeast Asia, Australia, South Africa and other waters, is an important marine economic crab in my country, and is also an important export-earning species. In recent years, the seawater crab breeding industry in my country has developed rapidly. In 2006, the breeding area of Scylla serrata and Portunus reached 762.77 million hectares, accounting for more than 95% of the breeding area of seawater crabs. The total output of both reached about 220,000 tons. Among them, the outpu...
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