siRNA of schistosoma japonicum katsurada SjFrzb2 gene and application thereof
A technology for schistosomiasis and schistosomiasis, applied in the fields of molecular biology and biomedicine, can solve the problems of non-specific and comprehensive gene expression inhibition and apoptosis of cells
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Embodiment 1
[0018] Example 1: In vivo screening of small RNAs
[0019] 1. Method steps
[0020] 1.1 siRNA molecules and SjFrzb2 Preparation of primers for quantitative real-time PCR of genes
[0021] Find the Schistosoma Frzb2 gene sequence (accession number AAX24900.2) on the NCBI website (http: / / www.ncbi.nlm.nih.gov / ), design and synthesize three pairs of dsRNA, the design principles mainly include the following aspects: 1. 1. Starting from the AUG start codon of the transcript (mRNA), look for the "AA or NA" double-linked sequence, and write down the 19 bases at the 3' end to design; 2. Avoid starting codons or nonsense regions Select the target sequence nearby; 3. The GC content of the sequence should be about 30%-60%; 4. Compare the selected sequence in the public database to ensure that the target sequence has no homology with other genes. Simultaneously synthesize a pair of unrelated control dsRNA (NC siRNA).
[0022] S1 dsRNA: sense: 5'-GCGCACAGAUUGCAUAUAUTT-3'(SEQ ID NO.1) ...
Embodiment 2
[0041] Embodiment 2: RNA interference experiment
[0042] 1. Method steps
[0043] 1.1 dsRNA injection
[0044] The experiment consisted of control group, unrelated control group (NC group) and S1 dsRNA treatment group, with three mice in each group. 4 days, 8 days, 12 days, 16 days, 20 days, 24 days, 28 days, 32 days, 36 days and 40 days after infection, 200 µl PBS, 200 µl NC dsRNA (8 µg) DEPC water and 200 µl SjFrzb2 Gene S1 dsRNA (8 µg) DEPC water, 10 times in total.
[0045] 1.2 Liver eggs and miracidia count
[0046] Collect the liver and weigh it, add dechlorinated water, mix with a homogenizer, and set the volume to 20 mL, take 1 mL of liver homogenate, add an equal volume of 10% NaOH (W / V), and digest in a 56°C water bath After 2 h, mix well after digestion is complete, take three 50 uL samples, and count eggs under microscope. Take 4 mL of liver homogenate in a narrow-necked flat-bottomed flask, add dechlorinated water, and cover it with a thin layer of cotton at...
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