Interference sequence for inhibiting Cnr1 gene expression, shRNA lentiviral vector as well as construction method and application of shRNA lentiviral vector
A lentiviral vector, interfering sequence technology, applied in DNA/RNA fragments, applications, genetic engineering, etc., can solve the problems of inability to treat irritable bowel syndrome, lack of understanding, etc., and achieve the relief of chronic visceral pain and depression-like behavior. Effect
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Embodiment 1
[0059] Western Blot detection, including the following steps:
[0060] 1. Histoprotein sample preparation:
[0061] 1) Extract fresh tissue, put it into a pre-cooled 2ml EP tube, and mark it;
[0062] 2) Add an appropriate amount of pre-cooled RIPA lysate and PMSF enzyme inhibitor, and use a homogenizer to fully homogenize the tissue on ice;
[0063] 3) Centrifuge the homogenized tissue at 4°C and 12000rpm for 15 minutes, and take the supernatant;
[0064] 4) After measuring the protein concentration of the sample by the BCA method, balance it, add the protein loading buffer (5×) in proportion, shake and mix, boil for 5 minutes, and store in the refrigerator at -20°C for later use;
[0065] 2. Preparation of polyacrylamide gel:
[0066] 1) Prepare 10% separating gel and 5% stacking gel respectively according to the SDS-PAGE gel preparation kit produced by Beyontien;
[0067] 2) After adding the prepared separation gel, add absolute ethanol to cover the surface, wait for th...
Embodiment 2
[0079] RT-qPCR detection of CB1 mRNA expression:
[0080] The primers used are designed in Table 1 below;
[0081] Table 1 Primer design in RT-qPCR detection
[0082] RatCnr1F 5'-CACCACAGACCTCCTCTACGT-3' RatCnr1R 5'-CATCTTTTTCTTGGAAGGGACTAC-3' Ratactin F 5'-TGAGAGGGAAATCGTGCGTG-3' RatactinR 5'-AGGGAGGAAGAGGATGCGG-3'
[0083] Extraction of total RNA:
[0084] 1) Collect fresh tissue with a pre-cooled 1.5ml RNase free centrifuge tube, add 450ul Buffer Rlysis-AG, fully homogenize with a homogenizer, and let it stand for 3 minutes;
[0085] 2) 12000rpm, 4°C, centrifuge for 3min, transfer the supernatant to a new 1.5ml centrifuge tube, add absolute ethanol half the volume of the supernatant, and mix well;
[0086] 3) Put the adsorption column into the collection tube, transfer all the solution to the adsorption column, let it stand for 1min, room temperature, 12000rpm, centrifuge for 1min, and pour off the waste liquid;
[0087] 4) Put the a...
Embodiment 3
[0104] Visceral pain threshold test, the specific steps are as follows:
[0105] (1) Apply paraffin oil to the uninflated balloon and place it in the colorectum. The end of the balloon is 0.5cm away from the anal verge and fixed with adhesive tape;
[0106] (2) Connect the catheter to the syringe and the sphygmomanometer through a three-way connection, place the rat on a clean table for observation, and start the experiment after about 15 minutes;
[0107] (3) The pressure starts from 0, and the pressure is increased at a constant speed. Observe the minimum pressure value that causes the lower abdominal wall to shrink and straighten or lift off the table, and record it as the pain threshold; each pressure is measured 3 times with an interval of 4 minutes, and the average value is taken;
[0108] The result is as image 3 As shown, compared with the virus control group injected with shRNA, the visceral pain threshold of shCB1-1 and shCB1-2 was significantly increased, indicati...
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