阅读说明：本技术 一种突变基因及其用于构建斑色鱼鳞癣小型猪模型的用途 (Mutant gene and application thereof in constructing speckled ichthyosis miniature pig model ) 是由 赵建国 王霄 曹春伟 海棠 周琪 王红梅 张颖 贾启涛 郑千涛 于 2018-08-21 设计创作，主要内容包括：本发明提供一种小型猪的ABCA12突变基因,所述突变基因与野生型猪ABCA12基因相比,具有IVS49-727A>G的突变。本发明提供了一种构建体,所述构建体包含所述ABCA12突变基因。本发明还提供了一种重组细胞,所述重组细胞由所述构建体转化受体细胞获得。本发明提供了一种制备人类斑色鱼鳞癣小型猪模型的方法,所述方法包括：改变小型猪的ABCA12基因,使ABCA12基因的位于第49号内含子的第727位碱基由A变为G。利用本发明提供的ABCA12突变基因制备大型人类斑色鱼鳞癣猪模型,研究人类斑色鱼鳞癣的发病机理,对于临床预防、诊断和治疗人类斑色鱼鳞癣具有重大的指导意义。(The invention provides a mini-pig ABCA12 mutant gene, which has IVS49-727A > G mutation compared with a wild pig ABCA12 gene. The invention provides a construct comprising the ABCA12 mutant gene. The invention also provides a recombinant cell obtained by transforming a receptor cell with the construct. The invention provides a method for preparing a human ichthyosis small pig model, which comprises the following steps: the ABCA12 gene of miniature pig is changed to change the 727 site base of No. 49 intron of ABCA12 gene from A to G. The ABCA12 mutant gene provided by the invention is used for preparing a large human ichthyophthiriasis pig model, researching the pathogenesis of the human ichthyophthiriasis, and has great guiding significance for clinically preventing, diagnosing and treating the human ichthyophthiriasis.)

1. A mini-pig ABCA12 mutant gene having a mutation of IVS49-727A > G compared to the wild type pig ABCA12 gene;

preferably, the sequence of the 49 intron of the ABCA12 mutant gene is shown in SEQ ID NO: 109;

preferably, the miniature pig is a bama miniature pig.

2. A construct comprising the ABCA12 mutant gene of claim 1.

3. A recombinant cell obtained by transforming a recipient cell with the construct of claim 2;

preferably, the recombinant cell is a porcine cell, more preferably a bama miniature porcine cell.

4. A method of making a human ichthyosis vulgaris miniature pig model, the method comprising:

the ABCA12 gene of miniature pig is changed to change the 727 site base of No. 49 intron of ABCA12 gene from A to G.

5. A method of screening a human ichthyosis versicolor mini pig model for ABCA12 gene mutation, 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) a sequence of the nucleic acid, or a complement thereof, having an IVS49-727A > G mutation compared to the wild-type ABCA12 gene, the mutation being indicative of human harlequin ichthyosis;

the biological sample is selected from at least one of blood, skin, hair, and muscle.

6. The method according to claim 5, wherein in step 2), determining the sequence of the nucleic acid comprises the steps of:

carrying out PCR by using DNA as a template and a specific primer of a pig ABCA12 gene to obtain an amplification product and sequencing the amplification product;

preferably, the sequence of the forward primer is shown as SEQ ID NO. 107, and the sequence of the reverse primer is shown as SEQ ID NO. 108.

7. A kit for screening a human ichthyosis variegata miniature pig model with ABCA12 gene mutation is characterized by comprising a specific primer of ABCA12 gene.

8. Use of the mutant gene of claim 1, the construct of claim 2, the recombinant cell of claim 3, or the kit of claim 7 in the preparation of a medicament for screening an animal model for the treatment and/or prevention of ichthyosis versicolor in humans; preferably, the animal model is a mammalian model; more preferably, the mammal is a mouse, monkey or miniature pig.

Technical Field

The invention belongs to the field of genetic engineering, and particularly relates to an ABCA12 mutant gene. The invention also relates to application of the mutant gene in constructing a speckled ichthyosis miniature pig model and a construction method of the speckled ichthyosis miniature pig model.

Background

The Guangxi Bama miniature pig has stable heredity, good consistency of phenotypic characteristics, mild temperament, small body size and various organ systems very similar to human bodies, and is suitable for medical and biological experiment application.

Ichthyosis punctatus (Harlequin ichthyosis) is an inherited skin disease and is one of the most serious autosomal recessive congenital ichthyosis. Diseased newborns are born with increased skin cornified layer thickness, full body coverage of armor-like scales, and often accompanied by abnormal facial features including blepharospectrum, labial eversion, deformed ears and nose.

Ichthyosis is caused by loss-of-function mutations in the ATP-binding cassette transporter a12(ATP binding cassette a12, ABCA12) gene. ABCA12 protein is localized in lamellar granules of epidermal keratinocytes and functions are associated with lipid transport into the extracellular space. Thus, the ABCA12 gene plays an important role in lipid transport in skin cells, intercellular lipid layer formation, and formation of the cell epidermal lipid barrier function.

The existing spot-color ichthyosis model is a mouse model. These mouse models can mimic a portion of the disease phenotype and help study pathological mechanisms, but suffer from several deficiencies. The skin structure of mice is different from that of humans, and therefore, there are some limitations in studying the pathology of diseases. The ichthyosis maculatus mice die after birth for several hours from dehydration, which makes them unable to become an ideal model for studying therapeutic methods and screening therapeutic drugs. Therefore, it is highly desirable to construct a model of the disease in a large animal having a physiological structure close to that of human, and to provide support for pathological studies, drug screening, drug efficacy evaluation, and the like for the disease.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides the ABCA12 mutant gene of the miniature pig, the phenotype and the genetic pattern of the transgenic family miniature pig with the mutant gene are consistent with the ichthyosis versicolor of the human, and the gene can be used as a large animal model of the human genetic disease, thereby providing support for pathological research, drug screening, drug effect evaluation and other researches aiming at the disease.

In one aspect, the present invention provides a mini-pig ABCA12 mutant gene having a mutation from a to G at base 727 of intron 49 (mutation of IVS49-727A > G) compared to the wild-type pig ABCA12 gene.

Preferably, the sequence of the 49 intron of the ABCA12 mutant gene is shown in SEQ ID NO: 109:

GTAAATAGAGTGCGGGAACTTTAATCAGATGACACAATGTTAGGATGATAGACGGGGAAAGGTAATCCTGTGCAATGTACATCGATTATGAATGTCCGCAATTGAACTATTTTGTTCTCATAGTCATGGGAGCAAGTCAGAAAGGTACCGTGGTGAGACTCCTGCTCCTCTGGTGGAACAGGGCCTAAGCAATCTTTTAAAAAACAAATTTCTGGTGACTTATGGCCCTCCTAACTCAACTACGACTCCCTATTACCTATCACCCATAGTTCAGCATCCCTCAGCTTAGGATGCAAGGCCTTTTATTATGTGGCCTCATCTGAATTTCCCGTATTTTTCACTACGAAACCCTCTCTTATCAGTCAGTCTGAGCGACTCCCAGTTCTACAGACACTCACTGCCCCCTCGCATCACCCTGCCCTTTCTTCAGCTGTGCTTTCTCCTGGAGTTGGTCTTTTCACAAACTCTACAATTTCTAATCCTACTAGTCCTTTAAGAAATAGTTCCTCCATGAAGTAGGCCCTTCCAAACCATTAAGTAAAAAAGCTACCAAAGCATCTTCTTTCTTTTTTTTCTTTTTTTGGCCGCCCCCAGGCGGGCCATGGACCAGATCCAAGCTGCAATTATGGCAACAAAGGATCCTTTAACCCGTTGCAGAGATGCTGCTGATCCTGTTGGAGCACAGCAGGAATTCTACTACTAATGCATTTTATCTGTGCCTTTTTAATAAGTTGACCTTGAAAGGTTCATCCTTTCAGTATAATTATCATATAATATTAAAAGGACTAGATATGTTTAGAATCAAGAGACTTGTTTACATCCTATATCCTAGCCTTACCCTAGCACTCTGTATATCCTGAAGCCAATTATTTAATTTCCATTAACTTTCATATTAAGTGGACATGGTGATACCTAAGATTGCCTTCAGTTATCATATCATAGTCCAAGAAATTAGAATTATGTAATCAATTACAGGTTAACTAGTTACAATACAAAAAAAAAAAAAAAAAGCCAACCGAATATGGAGTATAAGGCATAATGAAAAAAGCCTAATAAGCCTAATATTATTTTGGAAACAAAATGTGCCAAAATGTAGTTCTTGAGTCAAAGTTTCAAAGCACTTACAAGAAATACTGTGATTGATAAAATGGGTTTTCCAGGTAAGAGAGATGGCCTCAAATTTGAGAAGAAACAGTCGTAAACTCTATGTGAAGACATTTAATTTACATTAAAAGATTTTTTTTGTTGTTCTGCAGGGGAAAAGACATTTTAGGGAGTTCAAACAGTATTTCCTAGGTGCCATAATGAAAAAGTTAAGCCTATGCTTGTGAACTTAAAACCCAGTTTTCTGTGCTCAGAATGCTGATGTGATTCTGTCTTTTCAG。

preferably, the miniature pig is a bama miniature pig.

In another aspect, the invention provides a construct comprising the ABCA12 mutant gene.

The invention also provides a recombinant cell obtained by transforming a receptor cell with the construct. Preferably, the recombinant cell is a porcine cell, more preferably a bama miniature porcine cell.

According to an embodiment of the invention, the recombinant animal cell has a nucleic acid sequence in its genome encoding mutant ABCA12, wherein protein translation of the mutant ABCA12 is terminated prematurely compared to wild-type ABCA 12.

In yet another aspect, the present invention provides a method of preparing a human ichthyosis variegata miniature pig model, the method comprising:

the ABCA12 gene of miniature pig is changed to change the 727 site base of No. 49 intron of ABCA12 gene from A to G.

In some embodiments of the invention, according to actual needs, the ABCA12 gene of a normal pig is changed by using a genetic engineering technology, so that the 727 base is changed from A to G, and the human ichthyosis vulgaris pig model is obtained. In other embodiments, genetic engineering techniques can be used to alter the corresponding sites of the ABCA12 gene in animals other than swine to obtain a desired large or small human ichthyophthiriasis animal model, such as a human ichthyophthiriasis monkey model, a human ichthyophthiriasis mouse model, and the like.

The invention also provides a method for screening the human ichthyosis versicolor miniature pig model with ABCA12 gene mutation, which comprises the following steps:

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

2) determining the sequence of the nucleic acid DNA;

3) a sequence of the nucleic acid, or a complement thereof, having an IVS49-727A > G mutation compared to the wild-type ABCA12 gene, the mutation being indicative of human harlequin ichthyosis;

the biological sample is selected from at least one of blood, skin, hair, and muscle.

Preferably, in step 2), determining the sequence of the nucleic acid comprises the steps of:

and (3) carrying out PCR by using DNA as a template and a specific primer of the porcine ABCA12 gene to obtain an amplification product, and sequencing the amplification product.

Preferably, the sequence of the forward primer (F) is shown as SEQ ID NO:107, and the sequence of the reverse primer (R) is shown as SEQ ID NO: 108.

SEQ ID NO:107 CAGTCAGTCTGAGCGACTCC，

SEQ ID NO:108 ACAGAGTGCTAGGGTAAGGCTA。

The invention also provides a kit for screening the ABCA12 gene mutation human ichthyosis versicolor miniature pig model, which comprises a liquid or powder specific primer of the pig ABCA12 gene. The kit may include other reagents required for PCR, such as buffers, dntps, polymerase; reagents and consumables required for recovering PCR products, such as a sol solution, a collection tube, a washing solution and the like, can also be included. The DNA of a sample to be detected is used as a template, the kit and the method for screening the ABCA12 gene mutation human ichthyophthirius variegatus pig are used for detection, the operation is simple and convenient, and a large number of samples can be rapidly identified.

The invention also provides the use of the mutant gene, the construct, the recombinant cell or the kit of the invention in the preparation of an animal model for screening and treating and/or preventing the ichthyosis versicolor of the human; preferably, the animal model is a mammalian model; more preferably, the mammal is a mouse, monkey or miniature pig.

In order to obtain a large animal model of the ichthyosis versicolor of the human beings, ENU (N-ethyl-N-nitrosourea) chemical mutagenesis is carried out on the small Bama pigs, and the wild male small pigs are injected with ENU to obtain G0 generation small pigs; mating the miniature pig with a wild type female miniature pig of the same breed to obtain a G1 generation miniature pig; mating the obtained G1 generation male miniature pig with a wild type female miniature pig of the same breed to obtain a G2 generation miniature pig; mating the G1 generation male miniature pig with the G2 generation female miniature pig to obtain the G3 generation miniature pig, and carrying out phenotype screening on the G3 generation miniature pig to obtain the pedigree miniature pig with the speckled ichthyosis phenotype.

The human ichthyosis versicolor trait inheritance pattern of the pedigree miniature pig conforms to the Mendelian inheritance law of autosomal monogenic recessive inheritance. The number of wild-type individuals and mutant phenotype individuals in the G3 generation were counted and aligned, since wild-type: the mutant is 37:13, the ratio is about 3:1, and the separation law of Mendelian recessive inheritance 3:1 is met; also, the ratio of female to male in the mutant was 22:28, close to 1:1, indicating that the mutant phenotype is independent of sex, thus determining that the mutant phenotype is autosomal recessive.

In one embodiment according to the present invention, the injected dose of the ENU is 50 to 100mg/kg, more preferably, the injected dose of the ENU is 60 to 70 mg/kg; further preferably, the injection dose of the ENU is 65 mg/kg.

In one embodiment according to the present invention, further comprising: and (3) detecting the semen quality of the wild type male miniature pig after ENU injection, and mating the wild type male miniature pig after injection with the wild type female miniature pig of the same variety after the semen quality of the wild type male miniature pig after injection returns to a normal level.

The phenotype screening of the domestic miniature pig with the variegated ichthyosis phenotype is realized by the determination of phenotype observation, Skin penetration test (Skin permeability assay) and percutaneous water loss Test (TEWL).

Phenotypic analysis results showed that the harlequin ichthyosis model pig had a dry, hard, cracked skin and had an eyelid eversion phenotype.

Through whole genome association analysis, the pathogenic gene of the human ichthyosis vulgaris miniature pig model is determined to be ABCA12 gene, 53 exons and exon boundaries of the ABCA12 gene are completely sequenced, and the result shows that the mutation site of the mutant gene is positioned in No. 49 intron, so that the premature termination of protein translation can be caused. In particular, the mutant gene results in a splice change in the mRNA, i.e. an insertion of 132 bases is introduced between exons 49 and 50, resulting in premature termination of protein translation. According to data retrieval, the ABCA12 gene is well conserved in mammals such as human, monkey, pig, cow, sheep, horse, cat, dog, rabbit, mouse, rat and the like, so that the ABCA12 mutant gene provided by the invention is used for preparing a large human ichthyophthiriasis pig model, researching pathogenesis of the human ichthyophthiriasis, and has great guiding significance for clinical prevention, diagnosis and treatment of the human ichthyophthiriasis

Drawings

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

FIG. 1 is a graph of the whole body phenotype of the human ichthyosiform parvus minipig model (Mu) and the wild-type Guangxi Bama minipig (WT) of the present invention;

FIG. 2A shows the genotype identification of wild-type Guangxi Bama miniature pigs;

FIG. 2B shows the genotype identification results of the miniature pig model of human ichthyosis thyophthirius, with the arrow indicating the mutation site of the coding region of the gene;

FIG. 3A is a skin phenotype of a wild-type Guangxi Bama miniature pig;

FIG. 3B is a skin phenotype of the human ichthyosis mare minipig model of the present invention.

FIG. 4 shows the expression level of ABCA12 protein in the skin of a small pig model of human ichthyosis thyophthirius;

FIG. 5 is a schematic diagram of the mRNA splicing changes caused by the mutations in the genome of the human ichthyophthirius minipig model of the present invention.

Detailed Description

The present invention is further described below in conjunction with the following figures and examples, it being understood that the examples are intended to further illustrate and explain the present invention, and are not intended to limit the present invention.

Guangxi Bama miniature pigs were purchased from third department of military medical university and bred in the northern big animal research base of animal research institute of Chinese academy of sciences.

ENU was purchased from Sigma (N8509 bulk package).