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Lentiviral overexpression virus vector as well as preparation method and application thereof

A viral vector, overexpression technology, applied in the directions of viruses/phages, botanical equipment and methods, biochemical equipment and methods, etc., can solve the problems of time-consuming, labor-intensive, poor repeatability, low accuracy, etc., and achieve accurate and specific detection results. High performance and high sensitivity

Pending Publication Date: 2020-12-04
西人马大周(深圳)医疗科技有限公司
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Problems solved by technology

However, the HPV16 target gene plasmid in the cell quality positive quality control prepared by this method is easy to be lost during the long-term passage of HeLa cells, and the preparation of this type of positive quality control needs to prepare the HPV16 target gene plasmid again and transfect it into HeLa cells. Cells, the long-term use of this type of positive quality control is obviously time-consuming and laborious. In addition, there are problems of specificity, low accuracy and poor repeatability.

Method used

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  • Lentiviral overexpression virus vector as well as preparation method and application thereof
  • Lentiviral overexpression virus vector as well as preparation method and application thereof
  • Lentiviral overexpression virus vector as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] The construction of embodiment 1 lentiviral overexpression vector

[0046] This embodiment provides a method for constructing a lentiviral overexpression vector, comprising the following steps:

[0047] 1. Synthesis of HPV16E6 / E7 gene

[0048] According to the HPV16 gene sequence (Taxonomy ID: 333760) information recorded in the NCBI database, screen and design the following primers to HPV16 (E6 / E7)-f and HPV16 (E6 / E7)-r, the above-mentioned primers are shown in Table 1 below, and then Genomes of clinical HPV16 positive samples of human papillomavirus were extracted using Genomic DNA Extraction Kit Universal Genomic DNA Kit (article number: CW2298S) of Kangwei Century Company, and the method was referred to the kit instructions. Use the extracted clinical HPV16 positive sample genome as a template to amplify the HPV16E6 / E7 gene by PCR. The reaction system is shown in Table 2 below. The reaction conditions and process are as follows: step 1, keep at 98°C for 15s; step 2...

Embodiment 2

[0090] Embodiment 2 Positive quality control product

[0091] The genomic DNA of Hela-HPV16-L1 / E6 / E7 cells obtained in Example 1 was extracted by the magnetic bead method to obtain a positive quality control product. For details, please refer to the Guangzhou MagPure Universal DNA LQ Kit Extraction Kit (Cat. No.: D6314- 01).

experiment example 1

[0092] The detection of experimental example 1 HPV virus

[0093] (1) The genomic DNA of Hela-HPV16-L1 / E6 / E7 cells, HEK293T-HPV16-L1 / E6 / E7 cells and Hela cells obtained in Example 1 was extracted by the magnetic bead method, and the specific operation was carried out according to Guangzhou Meiji MagPure Universal DNALQ Kit Extraction Kit (Cat. No.: D6314-01) was carried out according to the instructions, and the concentration was measured by nucleic acid electrophoresis and ultraviolet spectrophotometer, and frozen at -20°C after aliquoting. The results are shown in Table 9 below and Figure 6 shown.

[0094] Table 9 Nucleic acid concentration and OD value

[0095]

[0096]

[0097] (2) qPCR-probe method to detect the genomes of various types of cells: extract each sample according to the magnetic bead method in step (1) for PCR amplification reaction, and mix the qPCR reaction system according to the following table 10, and perform qPCR reaction for each system accord...

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Abstract

The invention belongs to the technical field of cervical cancer virus detection, and particularly relates to a lentiviral overexpression virus vector. The lentiviral overexpression virus vector is obtained by co-transfecting host cells with recombinant expression plasmids and a packaging system to obtain lentiviral particles and infecting cervical cancer cells with the lentiviral particles; and the recombinant expression plasmids are obtained by inserting nucleotide sequences of an HPV16L1 gene and an HPV16E6 / E7 gene into multiple cloning sites in a PLVX-IRES-ZsGreen1 vector. The lentiviral overexpression virus vector disclosed by the invention does not disappear along with increase of passage times of HeLa cells, can be massively propagated in a growth and proliferation manner of the Helastable expression strain cells after one-time preparation, can also be stored for a long time by a liquid nitrogen cryopreservation method of the HeLa stable expression strain cells, and can be usedas a positive quality control material for detecting HPV16 and HPV18 viruses at the same time.

Description

technical field [0001] The invention belongs to the technical field of cervical cancer virus detection, and in particular relates to a recombinant expression plasmid, a lentivirus overexpression virus vector, a preparation method and an application thereof. Background technique [0002] According to worldwide statistics, cervical cancer is the second leading cause of death among women. About 270,000 women die from cervical cancer every year, most of which are caused by human papillomavirus (HPV) infection. ), while long-term persistent infection of high-risk subtypes of HPV virus is the main cause of cervical cancer, while HPV16 and HPV18 are the most carcinogenic HPV virus subtypes, accounting for about 50% and 20% of global cervical cancer cases, HPV Detection of high-risk subtypes is very important for early screening and treatment of cervical cancer in women. The traditional early screening of female cervical cancer mainly relies on cytological examination. During the p...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N7/01C12N15/37C12N15/11G01N33/569G01N33/96
CPCC12N15/86C12N7/00C07K14/005G01N33/56983G01N33/96C12N2740/15021C12N2740/15043C12N2710/20022
Inventor 聂泳忠丘国富马福军余桂荣许万里
Owner 西人马大周(深圳)医疗科技有限公司
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