LTalpha humanized genome, vector and application

Abstract

The invention relates to the field of genomes, in particular to an LTalpha humanized genome, a vector and application. According to the application, the human LT alpha gene is used for replacing the LT alpha gene of the mouse, so that the humanized LT alpha mouse can better simulate human immune response.

Description

Technical Field

[0001] The present application relates to the field of genome, and in particular to a LTα humanized genome, vector and application. Background Art

[0002] Lymphotoxin-α (LTa) is a key member of the TNF (tumor necrosis factor) superfamily and is involved in regulating the formation of lymphoid tissue, the regulation of immune responses, and a variety of inflammatory processes. It plays a vital role in the development and maintenance of the immune system and has made important contributions to revealing the biological functions and disease mechanisms of LTa.

[0003] For the purpose of studying the immune system, the LTα genome has important research significance, as it can reveal the basic functions of LTα in organisms, simulate the occurrence mechanisms of autoimmune diseases and tumors, and thus promote the development of targeted therapeutic drugs and treatments.

[0004] The research on LTα is still in its infancy. The current research methods for LTα genes include gene knockout, conditional gene knockout, etc., which are mainly carried out in mammals. However, in the above methods, due to the significant differences between the animal immune system and the human immune system, there is a certain degree of uncertainty in the process of introducing the gene model. Summary of the invention

[0005] Based on the above problems, the present application provides an improved LTα humanized genome for conducting research on LTα-related pathologies. At the same time, the present application also relates to the application of the above LTα humanized genome.

[0006] First, the present application provides a LTα humanized genome, which comprises: (i) an endogenous Lta promoter in the genome of a non-human animal, and (ii) humanized Lta gene, The humanized Lta gene is operably linked to the endogenous Lta promoter.

[0007] Preferably, the humanized Lta gene comprises an amino acid sequence encoding at least 95% identical to the following sequence: Seq1: MTLLGRLHLLRVLGTPPVFLLGLLLALPLGAQGLPGVGLTPSAAQTARQHPKMHLAHSTLKPAAHLIGDPSKQNSLLWRANTDRAFLQDGFSLSNNSLLVPT SGIYFVYSQVVFSGKAYSPKATSSPLYLAHEVQLFSSQYPFHVPLLSSQKMVYPGLQEPWLHSMYHGAAFQLTQGDQLSTHTDGIPHLVLSPSTVFFGAFAL.

[0008] Further preferably, the humanized Lta gene sequence is as follows: Seq1: MTLLGRLHLLRVLGTPPVFLLGLLLALPLGAQGLPGVGLTPSAAQTARQHPKMHLAHSTLKPAAHLIGDPSKQNSLLWRANTDRAFLQDGFSLSNNSLLVPT SGIYFVYSQVVFSGKAYSPKATSSPLYLAHEVQLFSSQYPFHVPLLSSQKMVYPGLQEPWLHSMYHGAAFQLTQGDQLSTHTDGIPHLVLSPSTVFFGAFAL.

[0009] Further preferably, the non-human animal is a mouse.

[0010] Ltα is located on chromosome 17. The mouse signal peptide and the human signal peptide are only 62% similar, but the cleavage sites of the two are similar. Therefore, the mouse signal peptide is retained to increase the secretion of human Ltα in mouse cells. Mouse and human Ltα have only one protein subtype, and the similarity is 81%. The secreted mature protein will be completely humanized.

[0011] The humanized Ltα cDNA is inserted into exon 2c, and the mouse signal peptide and human mature protein are driven by the mouse endogenous promoter and exogenous poly A. The cDNA insertion site will affect the DNaseI protection area, but will not affect the core binding site, which will effectively reduce the risk of dysregulation of humanized LTα or adjacent gene expression.

[0012] In the second aspect, based on the above genome, the present application further provides a vector comprising the above LTα humanized genome.

[0013] Preferably, the vector is a plasmid.

[0014] Preferably, the vector comprises a resistance gene for screening, homology arms, LoxP sites, DTA toxin gene, RNA polymerase II promoter, multiple cloning sites and gene editing elements as well as the LTα humanized genome described in any one of the above.

[0015] In addition, the present application also includes the use of any of the above-mentioned LTα humanized genomes in simulating human immune responses.

[0016] Preferably, the application method is to introduce the vector into a non-human mammal for expression.

[0017] Preferably, the non-human mammal is a mouse.

[0018] In summary, the above scheme provides a genome sequence for immunology research, which replaces the mouse LTα gene with the human LTα gene, so that the humanized LTα mouse can better simulate the human immune response. LTα plays a vital role in the formation of lymphoid tissue and the regulation of immune cells such as T lymphocytes and B lymphocytes. In the humanized model, the interaction between LTα and human-specific receptors and cells can be studied in a controlled in vivo environment, so as to understand the human immune process more accurately, which has important prospects in improving drug development, clarifying disease mechanisms, etc., such as: 1. Further improve the accuracy of the model By combining technologies such as CRISPR / Cas9, the genome structure of humanized LTα mice can be precisely adjusted to further optimize their ability to simulate human diseases. This will help enhance the application value of the model in the fields of immune diseases, tumor immunity, etc.

[0019] 2. Combining multiple gene modification technologies Future humanized model development can combine multiple gene editing technologies, such as modifying LTα together with other related immune factors (such as TNF-α, IL-6, etc.) to more comprehensively simulate human immune responses. This will provide more opportunities for the development of multi-target therapies.

[0020] 3. Promote the development of precision medicine With the rise of precision medicine, humanized LTα mouse models will play an important role in the development of personalized treatments. By studying the differences in individual immune responses, researchers will be able to develop personalized treatment plans for different patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the vector diagram shown in Example 2. DETAILED DESCRIPTION

[0022] The scheme in this application is further explained through the following specific implementation methods.

[0023] Example 1: Sequence Construction Design the sequence shown in SEQ1: Seq1: MTLLGRLHLLRVLGTPPVFLLGLLLALPLGAQGLPGVGLTPSAAQTARQHPKMHLAHSTLKPAAHLIGDPSKQNSLLWRANTDRAFLQDGFSLSNNSLLVPT SGIYFVYSQVVFSGKAYSPKATSSPLYLAHEVQLFSSQYPFHVPLLSSQKMVYPGLQEPWLHSMYHGAAFQLTQGDQLSTHTDGIPHLVLSPSTVFFGAFAL.

[0024] Example 2: Construction of vector The plasmid vector used in this application is shown in FIG. Figure 1 As shown, specifically, the plasmid contains the following genome parts: Resistance selection element: The plasmid contains a resistance gene for cell selection. The resistance cassette is labeled "Resistancecassette NEO", where the NEO gene encodes neomycin resistance and is used for positive selection in cells. This resistance gene will be used to select successfully transfected cell clones through the antibiotic G418 during use.

[0025] Homology arms (LA and SA): "LA" and "SA" can be seen on both sides of the plasmid. These two parts are sequences homologous to the target gene and are used for homologous recombination. Through these homology arms, the plasmid can be integrated into the genome of the host cell, thereby achieving targeted gene knock-in or knock-out.

[0026] LoxP site: The figure shows a "LoxP" site, which is an important element of the Cre-loxP system. The LoxP site can be used to mediate gene deletion or insertion at a targeted site through the Cre recombinase. This system is often used to construct conditional gene knockout mouse models.

[0027] DTA Toxin Gene: The plasmid also contains the "DTA" toxin gene (diphtheria toxin A chain) for negative selection. The presence of DTA helps to improve the accuracy of the selection by eliminating cells that have not undergone recombination, ensuring that only cell clones with correct insertion or recombination are retained.

[0028] Start sites and termination signals: The plasmid contains multiple start sites and termination signals for regulating gene expression. "Pol 2 Prom" indicates the RNA polymerase II promoter, which is able to initiate transcription of downstream genes. In addition, the plasmid also contains the SV40 polyA signal for transcription termination, which ensures correct mRNA splicing and polyadenylation.

[0029] Gene fragment and intron structure: In the central part of the plasmid, "LTA-Human-Mature formCDS" is marked, which represents the CDS sequence encoding the mature protein of human LTα (lymphotoxin α). The surrounding exon 1, exon 2a, exon 2b and intron sequences show that this plasmid may involve complex gene expression regulation and is suitable for studying gene function.

[0030] Multiple cloning site and gene editing elements: Plasmids contain a multiple cloning site (MCS) for inserting different gene fragments. This element greatly facilitates the scalability of plasmids and allows the insertion of target genes as needed.

[0031] Example 3: Cell electroporation 100 µg of linearized plasmid was transfected into 108 C57Bl / 6 embryonic stem cells at 260 volts and 500 µF.

[0032] Positive selection was initiated 48 hours after electroporation by adding 200 µg / ml G418 (active ingredient 150 µg / ml, Life Technologies, Inc.). Resistant clones were isolated and expanded in 96-well plates. Duplicate plates of 96-well plates were made. Plates containing ES cell clones expanded on gelatin were genotyped by PCR and sequencing analysis, with the primers selected and the PCR expected sizes shown in Table 1.

[0033] Example 4, Microinjection Embryo Transfer The electroporated cells were injected into blastocysts of albino C57BL / 6 mice, which were then transplanted into the oviducts of recipient mother mice, resulting in male chimeras with significant ES cell contribution.

[0034] Example 5: Chimerism analysis of mice The above-mentioned mice were analyzed. After dissecting the mice and separating their spleens, they were experimentally measured using a commercially available mouse lymphotoxin alpha (LTA) enzyme-linked immunosorbent assay kit. The expression of LTα was detected in both parents of the mice, proving that the above-mentioned experimental process had a good chimerism rate.

[0035] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. A LTα humanized genome, characterized in that: It contains: (i) an endogenous Lta promoter in the genome of a non-human animal, and (ii) humanized Lta gene, The humanized Lta gene is operably linked to the endogenous Lta promoter.

2. The LTα humanized genome according to claim 1, characterized in that: The humanized Lta gene comprises an amino acid sequence encoding at least 95% identical to the following sequence: Seq1: MTLLGRLHLLRVLGTPPVFLLGLLLALPLGAQGLPGVGLTPSAAQTARQHPKMHLAHSTLKPAAHLIGDPSKQNSLLWRANTDRAFLQDGFSLSNNSLLVPT SGIYFVYSQVVFSGKAYSPKATSSPLYLAHEVQLFSSQYPFHVPLLSSQKMVYPGLQEPWLHSMYHGAAFQLTQGDQLSTHTDGIPHLVLSPSTVFFGAFAL.

3. The LTα humanized genome according to claim 2, characterized in that: The humanized Lta gene sequence is as follows: Seq1: MTLLGRLHLLRVLGTPPVFLLGLLLALPLGAQGLPGVGLTPSAAQTARQHPKMHLAHSTLKPAAHLIGDPSKQNSLLWRANTDRAFLQDGFSLSNNSLLVPT SGIYFVYSQVVFSGKAYSPKATSSPLYLAHEVQLFSSQYPFHVPLLSSQKMVYPGLQEPWLHSMYHGAAFQLTQGDQLSTHTDGIPHLVLSPSTVFFGAFAL.

4. The LTα humanized genome according to claim 1, characterized in that: The non-human animal is a mouse.

5. A vector containing the LTα humanized genome according to any one of claims 1 to 4.

6. The carrier according to claim 5, characterized in that The vector is a plasmid.

7. The carrier according to claim 6, characterized in that The vector comprises a resistance gene for screening, homology arms, LoxP sites, a DTA toxin gene, an RNA polymerase II promoter, a multiple cloning site and a gene editing element, and the LTα humanized genome according to any one of claims 1 to 4.

8. Use of the LTα humanized genome according to any one of claims 1 to 4 in simulating human immune response.

9. The use according to claim 8, characterized in that: The vector according to any one of claims 5 to 7 is introduced into a non-human mammal to express the gene.

10. The use according to claim 9, characterized in that: The non-human mammal is a mouse.

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