New human-phosphoguanosine reductase, its coding sequence and application
A technology of guanosine phosphate and reductase, which is applied in the fields of biotechnology and medicine to achieve widespread effects
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Embodiment 1
[0092] Embodiment 1: Cloning of human GMPR2 cDNA
[0093] Total RNA of human dendritic cells was extracted with Trizol (Gibco). Then, poly(A) mRNA was isolated from total RNA. After the poly(A) mRNA was reverse-transcribed to form cDNA, the cDNA fragment was directional inserted into the multiple cloning site of the vector using the SuperScriptII cloning kit (purchased from Gibco), and transformed into DH5α bacteria to form a cDNA plasmid library. The 5' termini of randomly selected clones were sequenced by the dideoxy method. Comparing the determined cDNA sequence with the existing public DNA sequence database, it was found that the DNA sequence of one cDNA clone was a new full-length cDNA. The DNA sequence contained in the new clone is bidirectionally determined by synthesizing a series of primers. Computer analysis showed that the full-length cDNA contained in the clone was a new cDNA sequence (shown in SEQ ID NO: 1 and Figure 1), encoding a new protein (shown in SEQ ID ...
Embodiment 2
[0096] Embodiment 2: clone the coding sequence of human GMPR2 with RT-PCR method
[0097] Use Trizol (Gibco company) to extract the total RNA of B lymphoma cell line Raji cells in the logarithmic growth phase, take 6 mg of total cell RNA and 0.5 μg Oligo-dT 12-18 Mix and perform reverse transcription. The reverse transcription system is 20μl, add 80μl ddH after the reaction 2 O for dilution. The primers used in PCR amplification are as follows: sense primer 5'-G GAATTC CGCCATCATGCCTCATATTGACAACGATG-3' (SEQ ID NO: 3), antisense primer 5'-G GAATTC GTCGACGCACGCCTCACTGAAGATTG-3' (SEQ ID NO: 4). The PCR reaction volume was 50 μl, which contained 10 μl of reverse transcription template, 0.4 mM primers, 0.2 mM dNTP and 1U ExTaq DNA polymerase (Takara Inc.), and the amplification parameters were 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 45 seconds. Seconds, after 25 cycles, the PCR product was initially confirmed by 2% agarose gel electrophoresis. The result of DN...
Embodiment 3
[0098] Example 3 Northern blot analysis of GMPR2
[0099] Perform Northern blotting according to the following conventional method: put the filter membrane to be tested in 10ml of hybridization solution preheated at 68°C, and pre-hybridize at 68°C in a hybridization oven (Bellco) for 30 minutes; put the labeled cDNA probe at 95-100 Denature at ℃ for 2-5 minutes, put it on ice to cool quickly, and then add hybridization solution (the final concentration of cDNA probe is 2-10ng / ml or 1-2×10 6 cpm / ml), mix thoroughly, and hybridize at 68°C for 2 hours. After the hybridization, the filter membrane was washed several times with 2×SSC and 0.05% SDS at room temperature, followed by shaking and washing for 30-40 minutes, during which the washing solution was changed several times. Then rinse with 0.1×SSC and 0.1% SDS at 50° C. for 20 to 40 minutes with shaking. Finally, the filter membrane was wrapped with plastic cling film, and exposed to X-ray film at -70°C for 24 to 48 hours.
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