CN119685331A

CN119685331A - LT alpha humanized genome, vector and application

Info

Publication number: CN119685331A
Application number: CN202510179639.9A
Authority: CN
Inventors: 董源; 张欣欣; 柳雯昕
Original assignee: Shanghai Jinuowei Biotechnology Co ltd
Current assignee: Shanghai Jinuowei Biotechnology Co ltd
Priority date: 2025-02-19
Filing date: 2025-02-19
Publication date: 2025-03-25

Abstract

The present application relates to the field of genome, and in particular to a LTα humanized genome, vector and application. In the present application, by replacing the mouse LTα gene with the human LTα gene, the humanized LTα mouse can better simulate the human immune response.

Description

LT alpha humanized genome, vector and application

Technical Field

The application relates to the field of genome, in particular to an LT alpha humanized genome, a vector and application.

Background

Lymphotoxin-alpha (Lymphotoxin-alpha, LTa for short) is one of the key members of the TNF (tumor necrosis factor) superfamily, involved in regulating the formation of lymphoid tissues, the regulation of immune responses, and various inflammatory processes. It plays a critical role in the development and maintenance of the immune system, and has a significant contribution in revealing the biological functions and disease mechanisms of LTa.

In order to study the immune system, the LT alpha genome has important research significance, can reveal the basic function of LT alpha in organisms, simulate the occurrence mechanism of autoimmune diseases and tumors, and further promote the development of targeted therapeutic drugs and therapeutic methods.

In the research of the LT alpha, the research method of the LT alpha is in an initial state at present, and comprises gene knockout, conditional gene knockout and the like, which are mainly carried out in a mammal body, but in the method, due to the obvious difference between an immune system of the animal and an immune system of the human body, a certain uncertainty exists in the process of introducing a gene model.

Disclosure of Invention

Based on the above problems, the present application provides an improved LT alpha humanized genome for use in developing studies of LT alpha related pathologies. Meanwhile, the application also relates to application of the LT alpha humanized genome.

First, the present application provides an LT alpha humanized genome comprising:

(i) An endogenous Lta promoter of the genome of a non-human animal, and

(Ii) The Lta gene is humanized and the L gene is a humanized L gene,

Wherein the humanized Lta gene is operably linked to an endogenous Lta promoter.

Preferably, the humanized Lta gene comprises an amino acid sequence encoding at least 95% identical to the sequence:

Seq1:

MTLLGRLHLLRVLGTPPVFLLGLLLALPLGAQGLPGVGLTPSAAQTARQHPKMHLAHSTLKPAAHLIGDPSKQNSLLWRANTDRAFLQDGFSLSNNSLLVPTSGIYFVYSQVVFSGKAYSPKATSSPLYLAHEVQLFSSQYPFHVPLLSSQKMVYPGLQEPWLHSMYHGAAFQLTQGDQLSTHTDGIPHLVLSPSTVFFGAFAL.

further preferably, the humanized Lta gene sequence is as follows:

Seq1:

Further preferably, the non-human animal is a mouse.

Lt.alpha.is located on chromosome 17, the murine signal peptide and the human signal peptide are only 62% similar, but the cleavage sites of both are similar. Thus, the signal peptide of the mouse is preserved to increase the secretion amount of human Lt alpha in cells of murine origin. Lt.alpha.of murine and human origin only has 1 protein subtype and has a similarity of 81%. The secreted mature protein is fully humanized.

The cDNA of humanized Lt alpha is inserted into exon 2c, and the mouse-derived signal peptide and the human-derived mature protein are driven by the mouse endogenous promoter and exogenous polyA. The cDNA insertion site will affect the DNaseI protection region, but will not affect the core binding site, and will effectively reduce the risk of deregulation of expression of the humanized LT.alpha.or adjacent genes.

In a second aspect, the present application further provides a vector comprising the above-described lta humanized genome based on the above-described genome.

Preferably, the vector is a plasmid.

Preferably, the vector comprises a resistance gene, homology arm, loxP site, DTA toxin gene, RNA polymerase II promoter, multiple cloning site and gene editing element for selection, the LT alpha humanized genome of any one of the above.

In addition, the application also comprises the application of the LT alpha humanized genome in simulating human immune response.

Preferably, the method of use is to introduce the vector into a non-human mammal and express it.

Preferably, the non-human mammal is a mouse.

In summary, the above scheme provides a genomic sequence for studying immunology that is capable of better mimicking human immune response by substituting the human LT alpha gene for the mouse LT alpha gene. LT alpha plays a vital role in the formation of lymphoid tissues and the regulation of immune cells such as T-lymphocytes and B-lymphocytes. In the humanized model, the interaction of LT alpha with human specific receptors and cells can be studied in a controlled in vivo environment, so that the human immune process can be understood more precisely, and the method has important prospects in the fields of improving drug development, elucidation of disease mechanisms and the like, such as:

1. further improving the accuracy of the model

By combining CRISPR/Cas9 and other technologies, the genome structure of the humanized LT alpha mouse can be accurately regulated, and the capability of simulating human diseases is further optimized. The method is helpful for improving the application value of the model in the fields of immune diseases, tumor immunity and the like.

2. Combining multiple gene modification techniques

Future humanized model development may incorporate a variety of gene editing techniques, such as modification of LT alpha along with other related immune factors (e.g., TNF-alpha, IL-6, etc.) to more fully mimic human immune responses. This would provide more opportunities for the development of multi-target therapies.

3. Promote the accurate medical development

With the advent of accurate medicine, humanized LT alpha mouse models would play an important role in the development of personalized therapies. By studying the differences in individual immune responses, researchers can tailor personalized treatment regimens for different patients.

Drawings

FIG. 1 is a vector diagram shown in example 2.

Detailed Description

The embodiments of the present application will be further described in the following detailed description.

Example 1 sequence construction

Designing a sequence shown as SEQ 1:

Seq1:

EXAMPLE 2 construction of vectors

The vector diagram of the plasmid used in the present application is shown in FIG. 1, and specifically, the plasmid comprises the following genomic portions:

Resistance selection element plasmid contains resistance gene for cell selection. The resistance cassette is labeled "RESISTANCE CASSETTE NEO", wherein the NEO gene encodes neomycin resistance for positive selection in cells. This resistance gene will be used to select successfully transfected cell clones by the antibiotic G418.

Homology arms (LA and SA) the "LA" and "SA" are visible on both sides of the plasmid, which are sequences homologous to the target gene for homologous recombination. Through these homology arms, the plasmid can integrate into the genome of the host cell, thereby achieving targeted gene knock-in or knock-out.

LoxP site-the "LoxP" site is shown in the figure, which is an important element of the Cre-loxP system. The LoxP site can be used to mediate gene deletion or insertion of the targeting site by Cre recombinase. The system is commonly used for constructing a conditional gene knockout mouse model.

DTA toxin Gene the plasmid also contains a "DTA" toxin gene (diphtheria toxin A chain) for negative selection. The presence of DTA helps to improve the accuracy of the screen by removing cells that have not recombined, ensuring that only correctly inserted or recombined cell clones remain.

Initiation sites and termination signals the plasmid contains a plurality of initiation sites and termination signals for regulating gene expression. "Pol 2 Prom" means an RNA polymerase II promoter capable of initiating transcription of a downstream gene. In addition, the plasmid also contains an SV40 polyA signal for transcription termination, which ensures proper mRNA splicing and polyadenylation.

The gene fragment and intron structure are labeled "LTA-Human-Material form CDS" in the central part of the plasmid, which represents herein the CDS sequence encoding the Mature protein of Human LT alpha (lymphotoxin alpha). The surrounding exon 1, exon 2a, exon 2b and intron sequences show that the plasmid may be involved in complex gene expression regulation and is suitable for studying gene function.

Multiple cloning sites and Gene editing elements plasmids contain a Multiple Cloning Site (MCS) for insertion of different gene fragments. The element provides great convenience for the expandability of the plasmid, and the target gene can be inserted according to the requirement.

Example 3 cell electrotransformation

The linearized vector was transfected into 108C 57Bl/6 embryonic stem cells at 100. Mu.g of linearized plasmid, 260 volts, 500. Mu.F.

After 48 hours of electrotransformation, positive selection was initiated by adding 200 μg/ml G418 (150 μg/ml active ingredient from Life Technologies, inc.). Drug resistant clones were isolated and amplified in 96-well plates. A replica plate of 96 well plate was fabricated. The well plate containing ES cell clones amplified on gelatin was genotyped by PCR and sequencing analysis, and the primers and PCR expected sizes were selected as shown in Table 1.

EXAMPLE 4 microinjection embryo transfer

The above electrotransformed cells were injected into the blasts of albino C57BL/6 mice, and the blasts were transplanted into the oviduct of recipient mice to produce male chimeras with significant ES cell contribution.

Example 5 chimeric Rate analysis of mice

After the mice are analyzed and dissected, spleen of the mice is separated, experimental determination can be carried out through a commercially available mouse lymphotoxin alpha (LTA) enzyme-linked immunosorbent assay kit, and expression of the LT alpha is detected in parents of the mice, so that the experimental process has better chimeric rate.

The present embodiment is only for explanation of the present application and is not to be construed as limiting the present application, and modifications to the present embodiment, which may not creatively contribute to the present application as required by those skilled in the art after reading the present specification, are all protected by patent laws within the scope of claims of the present application.

Claims

1. An LT alpha humanized genome, characterized in that it comprises:

(i) An endogenous Lta promoter of the genome of a non-human animal, and

(Ii) The Lta gene is humanized and the L gene is a humanized L gene,

2. The LT a humanized genome of claim 1, wherein the humanized Lta gene comprises an amino acid sequence encoding at least 95% identical to seq id no:

Seq1:

3. the LT a humanized genome according to claim 2, characterized in that the humanized Lta gene sequence is as follows:

Seq1:

4. The LT a humanized genome of claim 1, wherein the non-human animal is a mouse.

5. A vector comprising the LT alpha humanized genome according to any one of claims 1 to 4.

6. The vector of claim 5, wherein the vector is a plasmid.

7. The vector according to claim 6, wherein the vector comprises a resistance gene for screening, a homology arm, a LoxP site, a DTA toxin gene, an RNA polymerase II promoter, a multiple cloning site and a gene editing element, and the lta humanized genome according to any one of claims 1 to 4.

8. Use of the LT alpha humanized genome of any one of claims 1-4 for mimicking a human immune response.

9. The use according to claim 8, wherein the vector according to any one of claims 5 to 7 is introduced into a non-human mammal and expressed.

10. The use of claim 9, wherein the non-human mammal is a mouse.