Standard plasmid for detecting copy number of exogenous gene of recombinant CVA10 vaccine as well as preparation method and application of standard plasmid
A CVA10 and foreign gene technology is applied in the field of standard plasmids for detecting the copy number of exogenous genes of recombinant CVA10 vaccine, and achieves the effects of saving detection time, simple operation and shortening detection time.
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[0083] 1.2. Preparation of recombinant plasmids
[0084] 1.2.1. The tandem CVA10-MOX-P1-3CD product was obtained by PCR
[0085] 1. Using the Hansenula yeast genome as the template and MOX-F2 and MOX-R3 as primers, PCR amplification was performed to obtain the endogenous gene MOX (SEQ ID No.1);
[0086] ②. Using the recombinant CVA10 vaccine Hansenula genome as the template, and using CVA10-P1-F1 and CVA10-P1-R1, CVA10-3CD-F1 and CVA10-3CD-R1 as primers, PCR amplification was performed to obtain foreign genes P1 fragment (SEQ ID No. 2) and exogenous gene 3CD fragment (SEQ ID No. 3);
[0087] ③. Establish the PCR reaction system shown in Table 1 below. The PCR amplification in Steps ① and ② is performed according to the PCR reaction system in Table 1.
[0088] Table 1 PCR reaction system
[0089]
[0090] ④. The PCR amplification of endogenous gene MOX, exogenous gene P1 and exogenous gene 3CD was detected by 1% agarose electrophoresis. The sizes of the corresponding PCR ...
Embodiment 2
[0160] Example 2: The difference from Example 1 is that the cloning vector in this example is the pUC18 vector, and the competent cells are TOP10 competent cells.
Embodiment 3
[0161] Example 3: The difference from Example 1 is that the cloning vector in this example is the pUC18 vector, and the competent cells are JM109 competent cells.
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