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.

Pending Publication Date: 2022-05-27
BEIJING MINHAI BIOTECH
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AI Technical Summary

Problems solved by technology

[0008] However, in the actual operation process, since the specific number of CVA10 gene P1 and gene 3CD inserted into the Hansenula genome is uncertain, it is necessary to clarify the specific insertion of exogenous gene P1 and exogenous gene 3CD in the process of standard product construction. The quantity in the Hansenula genome, so it is difficult to directly use the exogenous gene P1 and exogenous gene 3CD to prepare corresponding standards

Method used

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  • Standard plasmid for detecting copy number of exogenous gene of recombinant CVA10 vaccine as well as preparation method and application of standard plasmid
  • Standard plasmid for detecting copy number of exogenous gene of recombinant CVA10 vaccine as well as preparation method and application of standard plasmid
  • Standard plasmid for detecting copy number of exogenous gene of recombinant CVA10 vaccine as well as preparation method and application of standard plasmid

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[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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Abstract

The invention provides a standard plasmid for detecting the copy number of an exogenous gene of a recombinant CVA10 vaccine as well as a preparation method and application of the standard plasmid. The standard plasmid is formed by connecting an endogenous gene MOX fragment SEQ ID No.1, an exogenous gene P1 fragment SEQ ID No.2 and an exogenous gene 3CD fragment SEQ ID No.3 in series in a cloning vector. According to the method, the copy number of the inserted exogenous gene is simply, conveniently, rapidly, absolutely quantitatively and accurately analyzed by constructing the standard plasmid and adopting a Taqman probe-fluorescent quantitative PCR method, so that the detection time for determining the copy number of the exogenous gene of the recombinant CVA10 vaccine is effectively shortened, repeated operation is facilitated, and the detection efficiency is improved. The method has a wide application prospect in the determination of the copy number of the recombinant CVA10 vaccine exogenous gene constructed based on a hansenula polymorpha platform and the verification of the genetic stability of the recombinant CVA10 vaccine exogenous gene.

Description

technical field [0001] The invention belongs to the technical field of biological products, and in particular relates to a standard plasmid for detecting the copy number of exogenous genes of recombinant CVA10 vaccine and its preparation method and application. Background technique [0002] Coxsackievirus A10 (CVA10) belongs to the genus Enterovirus of the family Picornaviridae (Picornaviride), and is one of the pathogens causing Hand-foot-and-mouth Disease (HFMD). Hand, foot and mouth disease is a Class C infectious disease in my country. Its transmission route is complex and highly contagious, and it is easy to cause outbreaks or epidemics. It is often accompanied by herpes or ulceration of the hands, feet and oral mucosa. A few cases may develop into meningitis, pulmonary edema, Circulatory disturbances and even death. [0003] Before 2008, the main pathogens of hand, foot and mouth disease were Enterovirus 71 (EV71) and Coxsackievirus A16 (Coxsackievirus A16, CVA16) alte...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12N15/70C12N15/66C12N15/41C12N15/31C12R1/93
CPCC12Q1/701C12Q1/6851C12N15/70C12N15/66C07K14/005C07K14/39C12Q2600/166C12N2770/32022C12Q2531/113C12Q2561/101C12Q2545/114C12Q2537/16Y02A50/30
Inventor 徐颖之张改梅李国顺赵丽丽谢学超肖霞肖海峰顾美荣刘建凯
Owner BEIJING MINHAI BIOTECH
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