CN113774128B

CN113774128B - Gja8 application of gene mutation site in preparation of product for diagnosing cataract disease

Info

Publication number: CN113774128B
Application number: CN202111124004.7A
Authority: CN
Inventors: 田小芸; 许剑锋; 尤金炜
Original assignee: Eastern Theater General Hospital of PLA
Current assignee: Eastern Theater General Hospital of PLA
Priority date: 2021-09-24
Filing date: 2021-09-24
Publication date: 2022-08-23
Anticipated expiration: 2041-09-24
Also published as: CN113774128A

Abstract

The invention discloses an application of Gja8 gene mutation sites in the preparation of products for diagnosing cataract diseases, wherein the gene mutation sites are c.732G > T on Gja8 gene, namely mutation of nucleotide at 732 th position of Gja8 gene from G to T, so that amino acid at 244 th position of Gja8 protein coded by the gene mutation sites is mutated from Val to Phe, the mutation sites are not reported at present, and the mutation is found in rat models with cataract diseases and does not exist in normal rat models, which shows that the mutation can be used for diagnosing cataract diseases or evaluating the risk of cataract diseases, and has better application prospect.

Description

Gja8 application of gene mutation site in preparation of product for diagnosing cataract disease

Technical Field

The invention belongs to the field of in vitro diagnosis, and particularly relates to application of Gja8 gene mutation sites in preparation of a product for diagnosing cataract diseases, more particularly to mutation of the 732 th nucleotide of a Gja8 gene from G to T in the Gja8 gene mutation sites.

Background

Cataract (Cataract) is the first blinding disease worldwide. According to the world health organization, about 4500 million people are blind, 50% of which are caused by cataracts (Hammond CJ, Snieder H, Spectror TD, et al. genetic and environmental factors in age-related nuclear targets in monozgenetic and dizgenetic twins [ J ]. N Engl J Med,2000,342(24): 1786-90.). When the aqueous humor component or the permeability of the lens capsule and metabolic disturbance are caused by various reasons, the lens protein is denatured, water gaps, vacuoles, cell epithelial proliferation and the like appear among fibers, and the transparent lens becomes turbid, namely cataract is formed. The pathogenic factors of cataract can be divided into two categories, namely genetic factors and non-genetic factors, and cataracts caused by genetic factors comprise congenital cataracts, age-related cataracts and the like; non-genetic factors cause cataract including trauma, metabolic disease complications, poisoning, radiation and other physicochemical factors. The pathological mechanism of cataract formation is still unclear and the mechanisms of formation of different types of cataracts differ.

At present, although many cataract studies are followed by patients with congenital cataract discovered by clinicians during treatment, the variety of human congenital cataracts is great, and the sample size of patients capable of follow-up study is limited, so there is a great need in the art for spontaneous heritable animal models related to cataract for related studies. Animal model acquisition is generally achieved in three ways: induced mutation, genetic engineering and spontaneous mutation. There are many uncertainties in inducing mutations; although the genetic engineering technology is widely used in the field of constructing animal models, certain limitations exist, pathogenic genes of a plurality of human diseases are unknown, animal models required for research cannot be obtained through gene knockout and other modes, even mouse animal models without similar expression of human diseases are replicated, for example, the pathogenic gene of human Lowe syndrome is OCRL1, and the model of knocking out the mouse gene OCRL1 has no clinical expression of the human diseases; spontaneous mutation is an unavoidable and well-complemented model building, and few model animals have spontaneous and very similar human diseases.

In addition, few reports about hereditary congenital cataract rats exist at present, and BALB/c BK-Cat cataract mice bred by thin Kan et al in eight hospitals at home are lost; only cataractous BALB/c mice from the cross-bred population were reported by Shanghai laboratory animal center, Chinese academy of sciences. The congenital homozygous cataract rat model is not reported at present, and the cultivation of a spontaneous heritable cataract rat animal model has very important significance for early diagnosis of cataract, research on the generation and development mechanism of cataract, drug screening and prevention and treatment measures. In view of this, the invention discloses a spontaneous cataract rat animal model and a construction method thereof, the model is very similar to human nuclear cataract, and can be used as a model for researching the occurrence and development mechanism, early intervention, gene therapy, drug therapy and drug screening research and development of the disease.

Disclosure of Invention

In order to make up for the technical blank existing in the field, the invention aims to provide an application of Gja8 gene mutation sites in preparing a product for diagnosing cataract diseases, wherein the Gja8 gene mutation sites are c.732G & gtT, namely mutation of nucleotide 732 of Gja8 gene from G to T leads to mutation of 244 th amino acid of Gja8 protein encoded by the gene from valine (Val) to phenylalanine (Phe), the mutation exists in spontaneous cataract rat models, and the mutation does not exist in normal rats, thereby indicating that the mutation can be used for diagnosing the risk of cataract diseases.

The above object of the present invention is achieved by the following technical solutions:

in a first aspect of the invention, a diagnostic marker for cataract disease is provided.

Further, the diagnostic markers include a mutant Gja8 gene, a mutant Gja8 protein;

the mutant Gja8 gene is Gja8 gene carrying gene mutation site c.732G & gtT;

the mutant Gja8 protein is Gja8 protein carrying protein mutation site p.Val244Phe.

Further, the diagnostic marker refers to a molecular indicator with specific biological and biochemical characteristics, which can be used to determine the presence or absence of a specific disease or condition and/or the severity of a specific disease or condition, in the specific embodiment of the present invention, the disease is cataract disease, the diagnostic marker is Gja8 gene carrying the mutation site c.732G > T, and/or Gja8 protein carrying the mutation site p.Val244Phe.

The second aspect of the invention provides the application of the reagent for detecting Gja8 gene mutant and/or Gja8 protein mutant in preparing products for diagnosing cataract diseases.

Furthermore, the mutation site of the Gja8 gene mutant is the mutation from G to T at the 732 th nucleotide of the Gja8 gene, and the mutation site of the Gja8 protein mutant is the mutation from Val to Phe at the 244 th amino acid of the Gja8 protein.

Further, the reagent comprises a primer for detecting a mutation site of Gja8 gene 732 th nucleotide from G to T and/or a specific antibody for detecting a mutation site of Gja8 protein 244 th amino acid from Val to Phe;

the sequence of the primer is shown as SEQ ID NO. 1-SEQ ID NO. 2.

Further, the reagent also comprises a conventional reagent in a PCR amplification reaction, and/or a reagent used in a DNA extraction process, and/or a reagent used in a DNA sequencing process.

Further, conventional reagents in the PCR amplification reaction include (but are not limited to): dNTP, PCR buffer solution, magnesium ions, Tap polymerase and the like.

Further, methods that can be used for detecting the mutation site of the Gja8 gene include (but are not limited to): PCR (polymerase chain reaction) combined with one-generation sequencing, gene mutation DNA probe hybridization using a marker, a method using restriction fragment length polymorphism, or a method using sequence-specific primers, and the like.

In the specific embodiment of the invention, the c.732G & gtT mutation exists on Gja8 gene by sequencing spontaneous cataract rat model, and further, the mutation exists in spontaneous cataract rat model and does not exist in rat model without cataract disease by experimental verification of large sample, which indicates that the mutation can be used for diagnosing cataract disease.

In a third aspect of the present invention, there is provided a kit for diagnosing cataract disease.

Further, the kit comprises an effective amount of reagents for detecting Gja8 gene mutants and/or Gja8 protein mutants;

preferably, the Gja8 gene mutant has a mutation site from G to T at the 732 th nucleotide of Gja8 gene;

preferably, the Gja8 protein mutant has a mutation site from Val to Phe at amino acid 244 of Gja8 protein;

preferably, the kit further comprises a container, instructions for use, a positive control, a negative control, a buffer, an adjuvant, or a solvent.

Further, the sample detected by the kit is derived from blood or tissue of the subject.

Further, the kit also comprises buffer solution, dNTP and polymerase;

further, the kit also comprises reagents and consumables required for recovering PCR products;

furthermore, the reagent and the consumable comprise a sol solution, a collecting pipe, a washing solution and the like, DNA of a sample to be detected is used as a template, the kit and the method for screening the rat model with Gja8 mutant genes are used for detection, the operation is simple and convenient, a large number of samples can be rapidly identified, and the application prospect is good.

In a fourth aspect, the present invention provides the use of a diagnostic marker according to the first aspect of the invention in the manufacture of a kit for detecting cataract disease.

Further, the diagnostic marker is a reagent for detecting the diagnostic marker of the first aspect of the present invention;

further, the reagent comprises a reagent for detecting Gja8 gene mutant and/or protein mutant;

further, the reagent comprises a primer for detecting a mutation site of the 732 th nucleotide of Gja8 gene from G to T and/or a specific antibody for detecting a mutation site of Gja8 protein from Val to Phe;

the sequence of the primer is shown as SEQ ID NO. 1-SEQ ID NO. 2.

The fifth aspect of the invention provides the application of the primers shown as SEQ ID NO. 1-SEQ ID NO. 2 in the preparation of a kit for diagnosing cataract diseases.

According to a sixth aspect of the invention there is provided the use of a diagnostic marker according to the first aspect of the invention in the preparation of an animal model of cataract.

Furthermore, in the specific embodiment of the invention, the invention discovers that the mutant site c.732g > T exists on Gja8 gene in all spontaneous cataract animal models through experimental verification of large samples, and the mutant site c.732g > T does not exist on Gja8 gene in animal models (healthy animal models) without cataract disease, which shows that the mutant site c.732g > T on Gja8 gene is closely related to the occurrence and development of cataract disease, the nucleotide at 732 th position of Gja8 gene is mutated from G to T by changing Gja8 gene of animal models, so as to construct the animal models of cataract disease, and the constructed animal models can be used for pathological research, drug screening and drug effect evaluation of cataract disease.

Further, the cataract rat model of the present invention includes a homozygous cataract rat model and a heterozygous cataract rat model.

The seventh aspect of the invention provides a method for constructing a cataract rat model.

Further, the method comprises the steps of: the Gja8 gene of rat is changed, and the 732 th nucleotide of Gja8 gene is mutated from G to T.

Furthermore, the gene Gja8 of rat can be changed by using gene engineering technology according to the actual experiment requirement.

Further, the genetic engineering technology, also called genetic engineering technology, transgenic technology, gene modification technology, is a technology for directly manipulating the genome of an organism using biotechnology for changing the genetic material of cells. Gene transfer of the same species and across species is included to produce improved or new organisms. The new genetic material may be inserted into the host genome by isolating and replicating the desired genetic material using molecular cloning techniques to produce DNA sequences, or by synthesizing DNA and then inserting into the host organism. Nucleases can be used to remove or "knock out" genes. Gene targeting is a different technique that uses homologous recombination to alter endogenous genes, and can be used to delete genes, remove exons, add genes, or introduce point mutations.

Furthermore, the gene engineering technology for changing the Gja8 gene of the rat refers to changing the Gja8 gene of the rat by adopting a gene point mutation technology, and the gene point mutation technology refers to a technology for replacing an exogenous mutation site with a specific site in a genome in a homologous end recombination (HDR) mode so as to achieve site-specific mutation of the gene and obtain stable inheritance. At present, the point mutation is realized mainly by transferring a CRISPR/Cas9 system and a Donor sequence into a cell simultaneously, and introducing a gene sequence containing a point mutation site on the Donor sequence at a Cas9 cleavage site through an intracellular homologous recombination repair mechanism.

The inventors of the present application found for the first time that a novel mutation site c.732G > T was present in Gja8 gene in spontaneous heritable cataract rat models, and also sequenced large sample cataract rat models, including 65 healthy SD rats, 22 heterozygous cataract rats (homozygous cataract rats with BN rat), and 22 homozygous cataract rats, and found empirically that the above mutation was not present in all healthy SD rats, while c.732G > T heterozygous (bimodal) was present in Gja8 gene in 22 heterozygous cataract rats, and c.2G > T mutant site was present in Gja8 gene in 22 homozygous cataract rats, and it was found that the above mutation was not present in 65 healthy rats, and the above mutation was present in 44 cataract rats, which could be used to distinguish whether 100% of the subjects had cataract disease, the mutation site c.732G & gtT on the Gja8 gene is shown to be a brand new diagnosis marker for cataract disease diagnosis, and can be used for diagnosing whether a subject suffers from cataract disease, and can also be used for pre-pregnancy early warning and evaluating the prognosis condition of cataract patients.

An eighth aspect of the invention provides a method of screening a rat model of cataract with a Gja8 mutant gene in a non-diagnostic destination.

Further, the method comprises the steps of:

(1) extracting nucleic acid DNA of a biological sample to be detected;

(2) determining the sequence of the nucleic acid DNA;

(3) the nucleic acid sequence of step (2) or a complement thereof, having a c.732g > T mutation compared to the wild-type Gja8 gene, said mutation being indicative of cataract disease;

preferably, the biological sample is selected from blood and/or tissue.

Compared with the prior art, the invention has the advantages and beneficial effects that:

the invention discovers that Gja8 gene mutation site c.732G is more than T in a rat model with cataract disease for the first time, and verifies the mutation sites through large-scale sequencing, and the result shows that Gja8 gene mutation site c.732G is more than T in both homozygous and heterozygous cataract disease animal models, but the mutation sites do not exist in healthy animal models, which indicates that the mutation sites of Gja8 gene can be used in the diagnosis and detection of cataract disease, namely, the diagnosis of cataract disease is realized by detecting the existence of the mutation sites.

Drawings

Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1 shows a schematic diagram of homozygous spontaneously cataractous rats constructed according to the present invention;

FIG. 2 shows a schematic of healthy wild-type SD rats;

FIG. 3 shows a schematic representation of a hybrid cataractous rat constructed according to the present invention;

FIG. 4 is a diagram showing the sequencing result of Gja8 gene of a healthy wild type SD rat;

FIG. 5 is a graph showing the sequencing results of Gja8 gene from homozygous spontaneously cataractous rats constructed according to the present invention and heterozygous cataractous rats from wild type rats;

FIG. 6 is a diagram showing the sequencing result of Gja8 gene of homozygous spontaneous cataract rats constructed by the present invention.

Detailed Description

The present invention is further illustrated below with reference to specific examples, which are intended to be illustrative only and are not to be construed as limiting the invention. As will be understood by those of ordinary skill in the art: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents. The following examples are examples of experimental methods not indicating specific conditions, and the detection is usually carried out according to conventional conditions or according to the conditions recommended by the manufacturers.

Example 1 construction of spontaneous cataract rat animal model

1. Experimental Material

The occasional cataract SD rats in the population are provided by a comparative medical subject of Nanjing general Hospital in the military region of Nanjing, the experimental rats are all raised in a barrier environment, the temperature is 20-26 ℃, the relative humidity is 50% -70%, the illumination and the light are alternated, the rats are fed with free food and water, and the experimental process accords with the ethical regulations of experimental animals.

2. Experimental method

The construction and cultivation method of the spontaneous cataract rat animal model in the embodiment comprises the following steps:

(1) breeding the accidental cataract rats in the group for more than 8 weeks, taking male and female pairs for propagation, and recording the proportion of the cataract rats in the offspring;

(2) selecting cataract rats in offspring for continuous propagation, and finding three phenotypes of cataract rats, cataract rats with small eyeball and small palpebral fissure rats and cataract-free rats in offspring;

(3) taking a cataract rat non-homonidal mating propagation and a cataract rat with a small eyeball and a small palpebral fissure to perform homonidal mating propagation, and continuously verifying the genetic rule of a cataract mutant gene and the reproductive performance of the rat;

(4) mating a cataract rat and a cataract rat with a small eyeball and a small palpebral fissure with an SD rat respectively, and continuously observing the genetic rule of mutant genes of the cataract rat;

(5) taking a cataract rat and a cataract rat with a small eyeball and a small palpebral fissure to mate and propagate, and continuously observing the genetic rule of a mutant gene of the cataract rat;

(6) according to the results obtained in the step (3), the step (4) and the step (5), the cataract rat mutant gene is proved to be inherited as autosomal dominant and in a heterozygous state, and the cataract is proved to be homozygous phenotype along with the small bulbar palpebral fissure;

(7) according to the results obtained in the steps (2), (3), (4), (5) and (6), carrying out seed retention and littermate mating and passage on the cataract rat with the small bulbar palpebral fissure;

(8) taking cataract and small eyeball small palpebral fissure rats to mate and passage according to a sibling brother and sister mode, and recording the birth date, the number born, the weaning date, the number weaned and the fetal number;

(9) young food appears when inbred to the 5 th generation, cataract and cataract-free phenotype appear in a single eye of the 6 th generation, and a long tooth mouse appears in the 7 th generation;

(10) continuously selecting rats with cataract on both eyes for mating, wherein the offspring is differentiated, the rats have cataract on both eyes, the cataract on the left eye or the right eye and the phenotype of cataract observed by naked eyes are multiple, and the rats with cataract on both eyes are fewer;

(11) backcrossing is carried out on the 11 th generation according to the results of the step (9) and the step (10), then the sibling brothers and sisters are continuously bred until the eyes of the 15 th generation are seriously infected and die, and all the following generations 14 and 13 die;

(12) according to the result of the step (11), selecting another one after the tenth generation to continue propagating and passaging with the full sibling brothers and sisters, wherein the rat with the cataract in the later generation has the cataract in a single eye and the rat without the cataract is observed by naked eyes, selecting the rat with the cataract in two eyes to be reserved for passage, and gradually screening until all the offspring are the rat with the cataract in two eyes, thereby obtaining the homozygous spontaneous cataract rat model;

the homozygous spontaneous cataract rat constructed by the invention is matched with a wild type BN rat to obtain a heterozygous cataract rat model;

the rat model obtained by the above construction method is spontaneous mutation, and both eyes of rat mice develop cataract from open eyes, and the cataract phenotype of the rat is very similar to that of human nuclear cataract. The homozygous cataract rat is mated with SD rat, BN rat, F344 rat and the like, 100% heterozygous cataract rat can be obtained, the rat has cataract phenotype after 20 days of birth, and the heterozygous state cataract rat model and the homozygous state cataract rat model can be used for researching the generation and development mechanism of cataract.

3. Results of the experiment

The results show that the present invention successfully constructs a homozygous spontaneous cataract rat animal model (see fig. 1), and the cataract phenotype of the rat model is very similar to that of human nuclear cataract, fig. 2 is a schematic diagram of a healthy wild type SD rat, and two eyes are normal, and fig. 3 is a constructed heterozygous cataract rat animal model, which also shows cataract.

Example 2 screening and verification of cataract-causing genes

1. Experiment reagent and equipment

(1) Reagent: rat tail genomic magnetic bead purification kit (Tiandi and), amplification enzyme Vazyme-P222(2 × Rapid Taq Master Mix), primers (universal organism), agarose (solibao), DNA marker (solibao);

(2) the instrument comprises the following steps: nucleic acid extraction and purification workstation (heaven and earth), pipetting workstation (hamilton), PCR instrument (ABI), electrophoresis instrument/bath (zenith), gel imager (zenith).

2. Experimental methods

In this example, the constructed homo cataract rat model is sequenced to find that a c.732G > T mutation site exists on Gja8 gene, and then the site is verified by a large sample, wherein the verification process of the large sample is as follows:

using the homozygous spontaneous homocataract rat model constructed in example 1, and its bred offspring (heterozygous cataract rat model) and healthy Wt wild type SD rat genome as templates, PCR amplifying and sequencing the Gja8 gene mutation site sequence obtained by screening, comparing with the homozygous and wild type gene sequences, and determining whether c.732g > T mutation exists in Gja8 gene of the above three types of rats (homozygous cataract rat, heterozygous cataract rat, healthy wild type SD rat) to cause protein dysfunction, the sample information is shown in table 1;

TABLE 1 sample information

(1) Selecting rat tail of 109 rats (healthy: disease 65: 44) described in table 1, extracting DNA from tail;

(2) designing an amplification primer: designing amplification and sequencing primers at Gja8 predicted mutation sites, wherein the specific primer information is shown in Table 2;

TABLE 2 primer sequence information

(3) Performing PCR amplification on the 109 samples, wherein the PCR amplification system is shown in Table 3;

TABLE 3PCR amplification System

Seg.	reaction component	Volume(μL)
			1	2*Rapid Taq Master Mix	12.5
2	ddH ₂ O	9.5
			3	Primer A(10pmol/μL)	1
4	Primer B(10pmol/μL)	1
			5	Template(≈10-100ng/μL)	1

(4) After the PCR system is constructed in the workstation, the system is put into a PCR instrument for amplification, and the procedure of the amplification reaction is shown in Table 4;

TABLE 4 procedure for PCR amplification reaction

(5) Taking out the PCR product to carry out agarose gel electrophoresis detection: electrophoresis was performed using a 2% gel at 140V and 400mA for 25 min.

3. Results of the experiment

The sequencing result of healthy SD rats is shown in figure 4, the sequencing result of heterozygous cataract rats is shown in figure 5, and the sequencing result of homozygous cataract rats is shown in figure 6, wherein the results show that no c.732G & gtT mutation site exists on Gja8 genes in 65 healthy rats, while the c.732G & gtT mutation site exists on Gja8 genes of 22 homozygous cataract rats constructed by the invention, all the 732 sites are homozygous (mutant), and the c.732G & gtT heterozygous (bimodal) exists on Gja8 genes of 22 heterozygous cataract rats, and the summary experiment result is shown in table 5;

the results show that the Gja8 genes of 44 animal models with cataract disease all have c.732G & gtT mutation, and the Gja8 genes of 65 healthy animal models do not have c.732G & gtT mutation, namely, the Gja8 gene mutation site c.732G & gtT can be used for 100% distinguishing healthy people from patients with cataract disease, and the AUC value of a test subject working characteristic curve (ROC curve) is 1, thus proving that the mutation site c.732G & gtT on the Gja8 gene has very good diagnosis efficiency and can be applied to diagnosis of cataract disease.

TABLE 5 results of experiments in healthy SD rats, homozygous cataractous rats, and heterozygous cataractous rats

The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.

Sequence listing

<110> general hospital of eastern war zone of China's liberation army

Application of <120> Gja8 gene mutation site in preparation of product for diagnosing cataract disease

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<160> 2

<170> SIPOSequenceListing 1.0

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

tgtatggttt ccgcatcctg c 21

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gttggacagc gtgcattgtt ctag 24

Claims

1. The application of a reagent for detecting Gja8 gene mutant in preparing a product for diagnosing cataract disease is characterized in that the mutation site of the Gja8 gene mutant is the mutation from G to T of the 732 th nucleotide of a wild Gja8 gene;

the Gene ID of the wild type Gja8 Gene in NCBI is 29601;

the reagent comprises a primer for detecting a mutation site of the 732 th nucleotide of Gja8 gene from G to T.

2. The use of claim 1, wherein the primer has a sequence as shown in SEQ ID NO 1-SEQ ID NO 2.

3. Use according to claim 1, wherein the reagents further comprise reagents used in PCR amplification reactions, and/or reagents used in DNA extraction processes, and/or reagents used in DNA sequencing processes.

4. A method for screening a rat model of cataract with Gja8 mutant gene in a non-diagnostic destination, the method comprising the steps of:

(1) extracting nucleic acid DNA of a biological sample to be detected;

(2) determining the sequence of the nucleic acid DNA;

(3) the sequence of the nucleic acid DNA described in step (2) or the complementary sequence thereof, having a c.732G > T mutation compared to the wild-type Gja8 gene, said mutation being indicative of a cataract disease;

the wild-type Gja8 Gene has a Gene ID of 29601 in NCBI.

5. The method of claim 4, wherein the biological sample is selected from blood or tissue.