A porcine acute diarrhea syndrome coronavirus artificial chromosome recombinant vector and its construction method and application
An artificial chromosome, acute diarrhea technology, applied in the direction of viruses/phages, viruses, vectors, etc., can solve the problems of poor stability, cumbersome and time-consuming, and achieve the effect of reducing the cost of sequencing, improving the efficiency of recombination and the success rate of screening
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Embodiment 1
[0080] Example 1 Construction of SADS-CoV full-length infectious clone
[0081] Primer design principles: when designing primers, preset NotI and ApaI restriction sites at both ends of the vector pBeloBAC11-cm, preset NotI and XbaI restriction sites at both ends of the vector pBeloBAC11-spect (F1-F6), and preset NotI and XbaI restriction sites at both ends of the vector pBeloBAC11-spect EcoRV restriction sites are preset at both ends of pBeloBAC11-spect (F7-F10), and EcoRV restriction sites are preset at both ends of vector p15A-amp (F11-F14). Homologous arms are designed between each amplification primer sequence. There is an overlap of 30~60bp between the primers, which is synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. The specific sequences of the primers are shown in SEQ ID NO.1~42. .
[0082] PCR amplification program: 94 ℃ for 5 min; 98 ℃ for 10 s, 45-60 ℃ for 5 s, 72 ℃ for 5-90 s (according to the size of the target fragment, extension speed 10 s / kb), set 32...
Embodiment 2
[0112] Example 2 Construction of helper plasmid expressing SADS-CoV structural protein N
[0113] When designing primers, design homology arms between each amplification primer sequence. There is a 30-60 bp overlap between the primers. The specific sequences of the primers are shown in SEQ ID NO. Co., Ltd. synthesis.
[0114] PCR amplification procedure: same as Example 1.
[0115] PCR amplification reaction system: PrimeSTAR Max DNA Polymearse 25 μL, upstream and downstream primers (10pmol / μL) 0.5 μL each, template 0.5 μL, add ddH 2 O make up to 50 μL.
[0116] Gel recovery of amplification products: Place each PCR product on a 1% agarose gel for electrophoresis, and set the voltage and time on the electrophoresis apparatus to 125 V for 30 min respectively. After electrophoresis, under the ultraviolet light of the gel imager, carefully cut the gel at the target fragment band and place it in a sterilized 2 mL EP tube. Follow the gel recovery kit (TIANGEN). , DP214-03) inst...
Embodiment 3
[0125] Example 3 Construction of Recombinant Plasmids Deleting Part or All of ORF3a or ORF7a or ORF7b
[0126] Primer design principle: When designing primers, preset AscI restriction sites at both ends of amp-ccdB. Homologous arms are designed between each amplification primer sequence, and there is an overlap of 30~60bp between the primers. The specific sequences of the primers are shown in SEQ ID NO.108~121, and they are synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. .
[0127] PCR amplification procedure: same as Example 1.
[0128] PCR amplification reaction system: PrimeSTAR Max DNA Polymearse 25 μL, upstream and downstream primers (10pmol / μL) 0.5 μL each, template 0.5 μL, add ddH 2 O make up to 50 μL.
[0129] Gel recovery of amplification products: Place each PCR product on a 1% agarose gel for electrophoresis, and set the voltage and time on the electrophoresis apparatus to 125 V for 30 min respectively. After electrophoresis, under the ultraviolet ligh...
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