Bacterium expression vector for identifying tumor new antigens and method for screening and identifying tumor new antigens

An expression vector and antigen technology, applied in the field of tumor detection, can solve the problems of non-tumor toxicity and lack of complete specificity of central tolerance, and achieve the effects of high cost, long experimental period and long design time.

Inactive Publication Date: 2019-11-26
中生康元生物科技(北京)有限公司
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  • Application Information

AI Technical Summary

Problems solved by technology

Tumor-associated antigens refer to proteins overexpressed in tumor cells, but also expressed in normal cells, such as epidermal growth factor HER2, telomerase reverse transcriptase, etc., which are highly expressed in tumors. However, these antigens have a certain degree of Central tolerance and lack of complete specificity, same risk of on-target, non-tumor toxicity

Method used

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  • Bacterium expression vector for identifying tumor new antigens and method for screening and identifying tumor new antigens
  • Bacterium expression vector for identifying tumor new antigens and method for screening and identifying tumor new antigens
  • Bacterium expression vector for identifying tumor new antigens and method for screening and identifying tumor new antigens

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] This example provides a method for constructing a bacterial expression vector. The method for constructing the carrier specifically includes the following steps:

[0073] (1) clone the hly gene fragment from Listeria species, and clone the hly gene fragment on the ZS91 intermediate expression vector;

[0074] (2) Insert the ccdB gene fragment on the ZS91 intermediate expression vector in step (1) to increase the efficiency of cloning the neoantigen DNA sequence into ZS91, and refer to the ZS91-hly-ccdB bacterial expression vector diagram after construction figure 1 As shown, the full length is 8242bp.

Embodiment 2

[0076] This example provides a method for testing the bacterial expression vector constructed in Example 1. Specifically include the following steps:

[0077] (1) Synthesis of 3×OVA 8 (EQLE SIINFEKL TEWQLE SIINFEKL TEWQLE SIINFEKL TEW) and CEF sequences. The amino acid sequence of CEF is shown in SEQ ID NO.11, and the DNA sequence of CEF is shown in SEQ ID NO.10.

[0078] (2) Using ELISA to detect the secretion of IL-2 in B3Z cells by 3×OVA8.

[0079] First, 3×OVA8 was induced to express. On the clone ZS91-hly-3×OVA8 carrying 3×OVA8, the ZS91 empty vector was used as a positive control, and the vector map of the constructed ZS91-hly-3OVA8 was referred to figure 2 As shown, the full length is 5942bp.

[0080] Then transform the constructed ZS91-hly-3OVA8 vector into competent bacteria BL21, pick a single clone and shake the bacteria, dilute the bacterial solution in 100mL LB (Amp) at 1:100, add a final concentration of 1mM IPTG, and induce at 30°C Expression; bacte...

Embodiment 3

[0084] This example provides a method for screening and identifying tumor neoantigens. Specifically include the following steps:

[0085] (1) Discovery and prediction of neoantigens in clinical tumor samples: Tumor tissues were used for whole exome and RNA sequencing to identify mutation sites and expression of mutated genes, using NetMHC, NetCTLpan and IEDB to predict neoantigens, and in vitro according to the prediction results The DNA sequence of the candidate tumor neoantigen was synthesized and cloned into the ZS91 bacterial expression vector constructed in Example 1, and the synthesized DNA sequence of the candidate tumor neoantigen was introduced into the ZS91 bacterial expression vector through Nde I and Kpn I restriction endonucleases.

[0086] (2) Induce expression of candidate tumor neoantigens in bacterial strain BL21.

[0087] (3) Using Elispot to detect the secretion of INF-γ in T lymphocytes.

[0088] The 3 × OVA8 construct was transformed into bacterial compe...

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Abstract

The invention discloses a bacterium expression vector for identifying tumor new antigens and a method for screening and identifying the tumor new antigens, and relates to the technical field of detection of tumors. The expression vector can stably express the tumor new antigens, and comprises a plurality of insertion genetic fragments having special functions, wherein the insertion genetic fragments comprise a first genetic fragment which can effectively improve the submission efficiency of the tumor antigens and a second genetic fragment which can effectively improve the cloning efficiency. The invention further provides a method for performing immunology function verification on the tumor new antigens in flux and further quickly and effectively screening the tumor new antigens.

Description

technical field [0001] The invention relates to the technical field of tumor detection, in particular to a bacterial expression vector for identifying tumor neoantigens and a method for screening and identifying tumor neoantigens. Background technique [0002] At present, cancer is the second leading cause of human death after cardiovascular and cerebrovascular diseases, and its morbidity and mortality are gradually increasing, which has brought serious burdens to people's health and social economic development. The pathogenesis of all cancers comes from gene mutations or chromosomal mutations caused by changes in the genomic DNA sequence of cancer cells (Stratton, M.R., 2009), so it is difficult to treat cancer. The means of treating cancer mainly involves surgery, radiotherapy, chemotherapy, endocrine therapy and biological therapy, etc. Among them, the recurrence rate of surgical resection is high, and some cancers such as leukemia cannot be treated by surgery; chemothera...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70G01N33/68
CPCC12N15/70G01N33/6866G01N33/6869G01N2333/55G01N2333/57
Inventor 程旭东
Owner 中生康元生物科技(北京)有限公司
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