A long-acting artesunate drug for preventing schistosomiasis infection and its preparation method
A technology of artesunate and schistosomiasis, applied in the directions of anti-infective drugs, drug combinations, pharmaceutical formulations, etc., can solve the problems of short half-life, inapplicable to schistosomiasis infection, unable to maintain effective drug concentration for a long time, etc., to prolong half-life, prevent Schistosomiasis infection, the effect of good application prospects
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Embodiment 1
[0039] Example 1: (AS) n -Preparation of GST-NSP conjugate
[0040] (a) Construction of GST-NSP recombinant expression plasmid: Using pET41a(+) as the expression vector plasmid, firstly digest the expression vector plasmid pET41a(+) with BamH1 and Sal1. The specific conditions are as follows, the expression vector plasmid pET41a(+ ), 10μL; BamH11μL, Sal11μL, enzyme digestion buffer 5μL; double-distilled water 33μL, total volume 50μL, react in a constant temperature water bath at 37°C for 3 hours, and recover linearized pET41a(+) plasmid DNA by agarose gel electrophoresis , The NSP / pET43.1a plasmid DNA containing the NSP gene fragment was subjected to the same double digestion, and the NSP gene fragment with a molecular weight of about 1368bp was recovered by agarose gel electrophoresis, such as figure 1 Shown.
[0041] Connect the recovered NSP gene fragment with the above-mentioned double-enzyme-cut expression plasmid pET41a(+) to construct a recombinant expression plasmid GST-NSP...
Embodiment 2
[0051] Example 2: Determination of coupling rate
[0052] Determination of AS, (AS) by ultraviolet scanning method n -The highest absorption peak of GST-NSP and GST-NSP. Use the highest absorption wavelength to measure AS, (AS) n -The absorbance value of GST-NSP and GST-NSP.
[0053] According to the formula:
[0054]
[0055] Value; Ca represents the mole percentage of AS in the conjugate; Cb represents the mole percentage of the carrier protein NSP in the conjugate)
[0056] The results show that AS, (AS) n -The highest absorption wavelengths of GST-NSP and GST-NSP are: 260nm, 270nm and 280nm. Such as Figure 4 Shown. This result indicates that the conjugate has its own unique light absorption peak (270nm), indicating that the conjugate was successfully prepared. After calculating the coupling ratio of AS is 506:1, that is, 506 AS molecules are coupled to a GST-NSP protein.
Embodiment 3
[0057] Example 3: (AS) n -GST-NSP coupling in vitro killing children's insects and its half-lethal dose
[0058] For Assay (AS) n -Whether the GST-NSP conjugate retains the insecticidal activity of AS, use the in vitro culture method to determine AS and (AS) respectively n -GST-NSP conjugate activity, the specific method is as follows:
[0059] 1) Take 30 positive Oncomelania snails of Schistosoma japonicum into a 100mL beaker, wash several times with clean water, put a nylon net on the cup mask, add dechlorinated water and cover the nylon net, place it at 25-28℃, light for about 2h, and escape Cercariae.
[0060] 2) After soaking the 25×25mm cover glass in alcohol, wash it with tap water and wipe it clean.
[0061] 3) Load the ice-bathed RPMI1640 basal medium in a 15mL conical glass centrifuge tube, and place it on ice for refrigeration. Break off the 25×25mm cover glass, use tweezers to grab the glass flakes of appropriate length and width (can enter the tube but not reach the bott...
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