Use of an rb1 mutant in neurodegenerative diseases

Abstract

The application discloses application of an RB1 mutant in a neurodegenerative disease. The application finds through experiments that juvenile fish and adult heterozygotes of a zrb1-KO mutant zebrafish (2 bases are deleted at positions 67 and 68 of the 2nd exon of an rb1 gene) exhibit motor and memory learning dysfunction, exhibit increased post-mitotic neuron apoptosis in the hindbrain, and have the characteristics of a neurodegenerative disease, and therefore, the zrb1-KO mutant zebrafish can be used to construct an animal model of a neurodegenerative disease. In addition, the application also statistically analyzes the mutation frequency and mutation type of RB1 of a neurodegenerative disease patient, and verifies the pathogenicity of R621S and L819V mutations of RB1 in neuron apoptosis through experiments, thereby providing an effective approach for diagnosis or treatment of a neurodegenerative disease.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to an application of an RB1 mutant in neurodegenerative diseases. BACKGROUND

[0002] A finding of a variant in a patient's genome that is not known to be associated with health status is referred to as a variant of uncertain significance (VUS). In many cases, these variants are so rare in the population that little information is available about them. More information is often needed to determine whether a variant is associated with a disease. This type of information can include broader population data, functional studies, and tracking the variant in other family members who do or do not have the same health condition.

[0003] The vast majority of new variants that emerge when examining an individual's genome cannot be definitively classified as disease-causing or not. They are referred to as variants of unknown or uncertain significance. The more information scientists have about the variants seen in a particular family, group, or population, the more accurate their classification of the variants becomes. The number of VUS in this population is lower than in other populations. Currently, the genome information for people of European ancestry is relatively extensive compared to other groups, resulting in more VUS in other groups, which makes it more difficult to identify variants that cause disease.

[0004] Neurodegenerative diseases are characterized by the progressive loss of specific neuronal populations. Apoptosis of specific neurons is associated with the pathogenesis of neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS). Studies on Rb1 knockout mice by researchers have shown that Rb1 deletion leads to massive apoptosis in the nervous system, lens and skeletal muscle. Another group of researchers found that hyperphosphorylation of RB1 is associated with neurodegenerative diseases. Hyperphosphorylation of RB1 has been detected in pathological tissues of AD patients, ALS patients and PD patients. Knockout of mouse Nrmt1 can lead to inactivation of RB1, ultimately inducing neurodegenerative diseases, while the AD-related gene presenilin 1 (PS1) can protect against late neuronal death by inhibiting the phosphorylation of RB1. Although hyperphosphorylation of RB1 is associated with neurodegenerative diseases, there is no relevant report on the research of RB1 gene mutation and VUS in neurodegenerative diseases.

[0005] Zebrafish in vivo studies can be ingeniously used to shorten the gap between genetic diagnosis and functional studies. Zebrafish models are compatible with the latest mutagenesis techniques, which facilitate deciphering the underlying pathological mechanisms caused by gene mutations, opening up new avenues for VUS functional characterization. Due to zebrafish in vitro fertilization, zebrafish embryos can be microinjected with in vitro transcribed mRNA at the single-cell stage to overexpress disease-related proteins. This technique has been used to express patient-specific gene mutations associated with neurological diseases, such as amyotrophic lateral sclerosis or small fiber neuropathy. Therefore, zebrafish can serve as an advantageous tool for validating human VUS. SUMMARY

[0006] The primary object of the present application is to overcome the shortcomings and deficiencies of the prior art, and to provide an application of mutant zebrafish in preparing an animal model of neurodegenerative diseases.

[0007] Another object of the present application is to provide an application of RB1 mutant in preparing a drug for diagnosing or treating neurodegenerative diseases.

[0008] The object of the present application is achieved by the following technical solutions:

[0009] An application of mutant zebrafish in preparing an animal model of neurodegenerative diseases, wherein the mutant zebrafish is a zrb1-KO mutant zebrafish.

[0010] The zrb1-KO mutant zebrafish is a mutant formed by deleting 2 bases at positions 67 and 68 of exon 2 of zebrafish rb1 gene, which produces a premature stop codon to make the protein translation terminate prematurely, so that the normal Rb1 protein cannot be expressed and the normal rb1 gene function is lost.

[0011] The juvenile zrb1-KO mutant zebrafish exhibits abnormal motor function.

[0012] The adult heterozygote (zrb1-KO + / - ) of the zrb1-KO mutant zebrafish exhibits memory and cognitive dysfunction.

[0013] The zrb1-KO mutant zebrafish exhibits increased apoptosis of hindbrain neurons, which in turn causes increased hindbrain microglia.

[0014] The phenotype of increased apoptosis of hindbrain neurons of the zrb1-KO mutant zebrafish can be partially rescued by mRNA of zebrafish rb1 gene.

[0015] The zrb1-KO mutant zebrafish is subjected to targeted gene knockout by TALEN (transcription activator-like (TAL) effector nucleases) technology.

[0016] The TALEN target sequence of the zebrafish rb1 gene is as follows:

[0017] The left arm recognition sequence is: CCAGTCCACTAACT.

[0018] The right arm recognition sequence is: CTCCCATATTCTCCAT.

[0019] The post-neuronal apoptosis of the zrb1-KO mutant zebrafish is post-mitotic neuron.

[0020] The Rb1 regulates post-neuronal apoptosis of post-mitotic neurons through the Kmt5b-Bcl2a / Caspase axis.

[0021] The application of the RB1 protein mutant in the preparation of drugs for diagnosing and / or treating neurodegenerative diseases, wherein the RB1 protein mutant is at least one of R621S and L819V.

[0022] The present application verifies the high-frequency mutation R621S and L819V VUS clinical site of RB1 in neurodegenerative patients, and finds that the site has an important role in neuronal apoptosis.

[0023] The general idea of the application is:

[0024] (1) We cooperate with Guangzhou KingMed Medical Laboratory Center, which provides us with all the patient information collected from 2017-2020, and we screen the information of neurodegenerative disease patients among them, then group the neurodegenerative disease patients according to the types of PD / AD / HD / ALS, find the information of patients with RB1 mutation in these patients, and finally summarize the information of RB1 mutation in all neurodegenerative disease patients, and analyze the mutation frequency and mutation site of RB1.

[0025] (2) The characteristics of neurodegenerative diseases are progressive cognitive dysfunction and behavioral abnormalities, and zrb1-KO zebrafish is used to perform zebrafish behavior and T maze experiments. In addition, the type of neurons affected by rb1 deletion and the neuronal apoptosis pathway are analyzed.

[0026] (3) AO staining is used to verify the effect of the mutation site of human RB1 on neuronal apoptosis in zrb1-KO mutant zebrafish.

[0027] The present application has the following advantages and effects relative to the prior art:

[0028] (1) The present application analyzes the detection results and histological follow-up results of patients with neurodegenerative diseases collected by the inspection center, and statistically analyzes the mutation frequency and mutation type of RB1 in neurodegenerative diseases, and performs behavioral analysis on rb1 mutant zebrafish. For the first time, it is found that 2-3% of patients with neurodegenerative diseases have RB1 mutations through clinical sequencing data analysis, and the high-frequency mutation R621S and L819V VUS of RB1 in neurodegenerative patients are verified, and it is found that the site has an important role in neuronal apoptosis. In addition, the present application first verifies the pathogenicity of R621S and L819V mutations of human RB1 in neuronal apoptosis.

[0029] (2) In the present application, the zrb1-KO mutant is constructed, which can achieve: ① New breakthrough: there is no effective zebrafish model of human neurodegenerative diseases; ② High throughput: each pair of zebrafish can produce hundreds of eggs per week for use; ③ Low damage: zebrafish behavior experiment has no additional damage to zebrafish; ④ Easy to operate: zebrafish behavior experiment can be directly video tracked, and then analyzed; ⑤ Low cost: the cost of zebrafish feeding and consumption is much lower than that of mice, and the average consumption per day per mouse is about 1 / 100 of that of mice.

[0030] (3) In the present application, rb1-deficient mutants exhibit significant hindbrain neuronal apoptosis and increased hindbrain microglial cell infiltration, which can be partially rescued by wild-type zrb1 mRNA; the apoptotic cells in zrb1-KO mutants are post-mitotic neurons in the hindbrain, and Rb1 regulates post-mitotic neuronal apoptosis independently of its effect on neural stem cell proliferation.

[0031] (4) In the present application, it is found that zrb1-KO mutant zebrafish larvae and Rb1 adult heterozygotes exhibit motor and memory learning dysfunction, and zrb1-KO mutant zebrafish exhibit increased hindbrain post-mitotic neuronal apoptosis, and the apoptosis is regulated through the kmt5b-bcl2 / caspase pathway, that is, Rb1 can combine Kmt5b to inhibit the expression of bcl2a / caspase, regulate the apoptosis pathway, and maintain the survival of post-mitotic neurons. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1is a mutation status chart of RB1 in patients with neurodegenerative diseases; wherein, A is the mutation rate of RB1 in the exome sequencing data of patients with neurodegenerative diseases provided by the Jin Yuan Medical Examination Center

PD (blood sample: 189), AD (blood sample: 33), HD (blood sample: 69), ALS (blood sample: 75)

[0033] Figure 2 is a chart of Rb1 deletion impairing the motor function of juvenile zebrafish and the memory and cognitive ability of adults; wherein, A is the knockout of zebrafish rb1 gene mediated by TALEN technology (in the zebrafish rb1 gene, the position and sequence of the TALEN target site are enlarged; the zrb1-KO mutant of zebrafish is a 2-base deletion on the 2nd exon of the zebrafish rb1 gene, which prematurely terminates protein translation; the truncated Rb1 protein is represented by a magenta diagonal line); B is the movement trajectory of siblings and zrb1-KO mutants within 5 min at 5 dpf; B' is a statistical chart of the movement distance of siblings and zrb1-KO mutants within 5 min in B (t-test; mean ± SEM; ***P < 0.001; n = 12); C is the T maze experiment for detecting adult wt and zrb1-KO + / - is a heat map of the movement trajectory of heterozygotes within 5 min before and after 7-day food-induced stimulation (right arm EC: eutrophication area); C' is the retention time of adult wt and zrb1-KO + / - is the retention time of heterozygotes in the left arm and the right arm before and after training (t-test; mean ± SEM; **P < 0.01; ns, not significant; n = 10).

[0034] Figure 3are the apoptosis situation diagrams of post-mitotic neurons after Rb1 deletion; wherein, A and A' are the laser confocal imaging and statistical diagram of the sibling and zrb1-KO mutant post-mitotic neurons in the hindbrain at 3 dpf (white arrows indicate the position of the apoptotic bodies) (t-test; mean ± SEM; ***P<0.001; n=8); B and B' are the results of neutral red (NR: label microglia cells) staining and the statistical results of microglia cells in the cerebellum and hindbrain of sibling and zrb1-KO mutant at 5 dpf (white dotted line indicates the position of the cerebellum and hindbrain) (t-test; mean ± SEM; ***P<0.001; n=10); C and C' are the results of acridine orange (AO) staining and statistical results of sibling and zrb1-KO mutant under the background of Tg(nbt:dsRed) at 3 dpf (white dotted line indicates the position of the cerebellum and hindbrain) (t-test; mean ± SEM; ***P<0.001; n=10); D and D' are the results of acridine orange (AO) staining and statistical results of sibling and zrb1-KO mutant under the background of Tg(nbt:dsRed) at 3 dpf, respectively injected with ddH20 and rb1 mRNA (white dotted line indicates the position of the cerebellum and hindbrain) (one-way ANOVA; mean ± SEM; ***P<0.001; n=10).

[0035] Figure 4 are the apoptosis situation diagrams of post-mitotic neurons regulated by Rb1; wherein, A is that the NSPCs population and post-mitotic neuronal cell population in the whole brain are sub-clustered into 21 groups, and then defined by marker genes (F: forebrain, M: midbrain, Mye: hindbrain, NSPCs: neural stem / progenitor cells); B and C are the top 20 pathways of differential genes in the hindbrain and cerebellum post-mitotic neurons of zrb1-KO mutant compared with sibling, which can be enriched in the apoptosis pathway (indicated by red arrow); D is the AO staining to detect cell apoptosis after microinjection of nestin:Cas9-T2A, mCherry, U6:gRNA(rb1) plasmid at the single cell stage of wild type zebrafish embryo (white arrow indicates the apoptotic cells); D' is the statistical diagram of AO staining in D (t-test; mean ± SEM; *** P<0.001; n≥10 samples per group); E is the AO staining to detect cell apoptosis after microinjection of huc:Cas9-T2A, mCherry, U6:gRNA(rb1) plasmid at the single cell stage of wild type zebrafish embryo (white arrow indicates the apoptotic cells); E' is the statistical diagram of AO staining in E (t-test; mean ± SEM;*** P<0.001; n≥10 per group).

[0036] Figure 5 is a diagram of the situation of Rb1 regulating post-mitotic neuronal apoptosis through Kmt5b-bcl2a / caspase pathway; wherein, A is to express bcl2a in sibling and zrb1-KO mutant nervous system, 3dpf AO staining is used to detect the number of apoptotic cells (white dotted line encloses the cerebellum and medulla region); A' is the statistical chart of AO staining in A (one-way ANOVA; mean±SEM; *** P<0.001; n≥10 per group); B is to use Z-VAD-FMK inhibitor and DMSO to treat zebrafish embryos in sibling and zrb1-KO mutant, 3dpf AO staining is used to detect the number of apoptotic cells (white dotted line encloses the cerebellum and medulla region); B' is the statistical chart of AO staining in B (one-way ANOVA; mean±SEM; *** P<0.001; n≥10 per group); C is the expression of kmt5b in neural stem progenitor cells (NSPCs), forebrain (F), midbrain (M), cerebellum (Ce), medulla (Mye) group and other groups; D is to microinject Huc:kmt5b-eGFP plasmid in sibling and zrb1-KO mutant, 3dpf Tunel staining is used to detect the number of apoptotic cells (white dotted line encloses the cerebellum and medulla region); D' is the statistical chart of Tunel + staining in D (one-way ANOVA; mean±SEM; *** P<0.001; n≥10 per group); E is to microinject kmt5b MO plasmid in sibling and zrb1-KO mutant, 3dpf Tunel staining is used to detect the number of apoptotic cells (white dotted line encloses the cerebellum and medulla region); E' is the statistical chart of Tunel + staining in E (one-way ANOVA; mean±SEM; *** P<0.001; n≥10 per group).

[0037] Figure 6 is a diagram of the correlation between R621S and L819V mutations of human RB1 protein and neuronal apoptosis; wherein, A is to inject ddH2O, hRB1 mRNA, hRB1 Arg621Ser mRNA or hRB1 Arg798TrpmRNA, and the number of apoptotic cells in A was counted (white dotted line to enclose the cerebellum and medulla oblongata position, one-way ANOVA; mean ± SEM; ***P < 0.001, ns, not significant; n≥10 samples per group). DETAILED DESCRIPTION

[0038] The application will be described in further detail below with reference to Examples, but the embodiments of the application are not limited thereto. The reagents, methods, and apparatuses employed in the present application are those conventionally used in the art unless otherwise specified. Each of the raw materials used in the following Examples is commercially available unless otherwise specified.

[0039] The term "sibling" or "sibling wild-type zebrafish" used in the present application refers to an individual descended from the same parents.

[0040] The term "dpf" used in the present application refers to the number of days after fertilization. For example, "3dpf" refers to 3 days after fertilization, and "5dpf" refers to 5 days after fertilization.

[0041] Example 1

[0042] 1. Analysis of data from patients with neurodegenerative diseases

[0043] Guangzhou Jin-Yu Medical Examination Center provided us with all the patient information they collected from 2017 to 2020 (with the consent of the patients), we screened the information of patients with neurodegenerative diseases from them, then grouped the patients with neurodegenerative diseases according to Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD) and amyotrophic lateral sclerosis (ALS), found the information of patients with RB1 mutations in these patients, and finally summarized the information of RB1 mutations in all patients with neurodegenerative diseases, and analyzed the mutation frequency and mutation sites of RB1.

[0044] The results are shown in Figure 1 Figure 1A). The frequency of RB1 mutations affecting protein structure was 2.64% in PD, 3.03% in AD, 2.85% in HD, and 2.66% in ALS (Figure 1A). These RB1 mutations included 6 sites: 4 missense mutations (G38D, R621S, R789W, L819V) and 2 splice mutations (X405_splice, X406_splice), and were found in 9 patients with neurodegenerative diseases (Figure 1B). Figure 1 Figure 1 ​B). The frequency of the four missense mutations in patients with neurodegenerative diseases was slightly higher than that in the ALAF (Allele Frequency Aggregator Project) project and the normal East Asian population, but only R621S and L819V gene mutations showed statistical significance Figure 1 C).

[0045] Note: "R621S" means the 621st arginine (R) in RB1 is mutated to serine (S), "L819V" means the 819th leucine (L) in RB1 is mutated to valine (V), and the others are the same. "X405_splice" means the mutation occurs in the intron of RB1, which occurs 3 bases after the 1215th position of the transcript mapped to the gene coordinates, and forms a stop codon at the 405th position. "X406_splice" means the mutation occurs in the intron of RB1, which occurs 8 bases before the 1216th position of the transcript mapped to the gene coordinates, and forms a stop codon at the 406th position.

[0046] 2. Breeding of zebrafish

[0047] The breeding of zebrafish in the present application is as described in the literature (Westerfield M: The zebrafish: guide for the laboratory use of zebrafish (Brachdanio rerio). Edition by Eugene, OR, M. Westerfield, 1993).

[0048] The following strains of zebrafish are used in the present application: AB wild-type zebrafish, zrb1-KO mutant zebrafish, and zrb1-KO mutant zebrafish with Tg(Xla.Tubb:DsRedx) (alias Tg(nbt:dsRed2)) and Tg(Xla.Tubb:bcl-2) (alias Tg(nbt:bcl2a)) transgenic backgrounds; wherein,

[0049] The zrb1-KO mutant zebrafish with Tg(Xla.Tubb:DsRedx) transgenic background is obtained by crossing adult zrb1-KO heterozygous zebrafish with transgenic Tg(Xla.Tubb:DsRedx) zebrafish; the zrb1-KO mutant zebrafish with Tg(Xla.Tubb:bcl-2) transgenic background is obtained by crossing adult zrb1-KO heterozygous zebrafish with transgenic Tg(Xla.Tubb:bcl-2) zebrafish;

[0050] The transgenic zebrafish Tg (Xla.Tubb: DsRedx) were obtained according to the reference (Sun, W., Wang, M., Zhao, J. et al. Sulindac selectively induces autophagic apoptosis of GABAergic neurons and alters motor behavior in zebrafish. Nat Commun 14, 5351 (2023).);

[0051] The transgenic zebrafish Tg (Xla.Tubb: bcl-2) was obtained according to the literature (Xu J, Wang T, Wu Y, Jin W, Wen Z. Microglia Colonization of Developing Zebrafish Midbrain Is Promoted by Apoptotic Neuron and Lysophosphatidylcholine. Dev Cell. 2016 Jul 25; 38(2): 214-22.).

[0052] 3. Construction of zrb1-KO mutant zebrafish

[0053] 3.1 TALEN knockout of the zebrafish rb1 gene

[0054] In this experiment, the TALEN knockout target was designed first, and then the target TALEN target left arm mRNA chain and right arm mRNA chain were synthesized in vitro. Then, they were mixed and microinjected into the embryos of newly born AB wild-type zebrafish. After PCR amplification verification, the remaining embryos with high efficiency were cultured and grown for F0 screening. After screening, the F0 was hybridized with wild-type zebrafish to obtain heterozygous mutants with mutated rb1 (zrb1-KO + / - heterozygote).

[0055] Figure 2 As shown in A, subsequent experiments mainly use mutants with two base deletions. The mutant zebrafish with two base deletions is a zebrafish rb1 gene exon 2 with two bases 67 and 68 (zrb1-KO, exon2: 67-68) missing, resulting in a stop codon at position 98 in the amino acid sequence, causing a frameshift during translation, resulting in premature termination, and the final translated protein lacking all important functional domains. The specific steps include the following:

[0056] 3.1.1 TALEN knockout target design

[0057] According to the information of ensembl website, the full length of zebrafish rb1 gene 001 transcript is 3862 bp, and the translated protein size is 903 aa (ENSDART00000024309.10). These amino acid sequences constitute four important domains, which are closely related to the function of Rb1 protein. There is only one transcript, based on which we design the TALEN recognition knockout target at the 2nd exon after the translation start site of the 1st exon. This site is before the expression site of the four protein domains. If the knockout is successful, it is most likely to cause the loss of Rb1 protein function.

[0058] We constructed the TALEN target sequence of zebrafish rb1 gene: the left arm recognition sequence is CCAGTCCACTAACT, and the repeat variable di-residues (RVDs) are HD HD NI NN NG HD HDNI HD NG NI NI HD NG HD HD NI NG; the right arm recognition sequence is CTCCCATATTCTCCAT, and the RVDs are HDNG HD HD HD NG NI NG NI NG NG HD NG HD HD NI NG.

[0059] The specific construction method is as follows:

[0060] 1) Single vector, the plasmid backbone for target construction is PMD18-T vector (Takara, 6011). There are four single vectors, pA, pT, pC and pG (NN), each corresponding to recognizing one nucleotide base.

[0061] 2) Assemble RVDs according to the sequence of the target site by single vector

[0062] ① Firstly, the pA vector at 5' end was double digested with NheI and HindIII, the system was placed in 37℃ incubator, and the enzyme was digested overnight. The next day, the enzyme product was mixed with loading buffer (TransGen, GH101-01), and 1% agarose gel was used for electrophoresis separation. The agarose gel block containing the uncut plasmid and the agarose gel block containing the completely cut fragments were separated under ultraviolet light, and the target fragment with a size of about 2.8 KB was obtained by gel cutting purification. The pA vector was obtained from the reference (Huang, Peng et al. "TALEN construction via "Unit Assembly" method and targeted genome modifications in zebrafish." Methods (San Diego, Calif.) vol. 69, 1 (2014): 67-75.).

[0063] ② The pT vector at 3' end was double digested with SpeI and HindIII, and was also placed in 37℃ incubator for overnight enzyme digestion. The loading buffer was mixed, and 2% agarose gel was used for electrophoresis. The band with a size of about 102 bp was purified by gel cutting. The pT vector was obtained from the reference (Huang, Peng et al. "TALEN construction via "Unit Assembly" method and targeted genome modifications in zebrafish." Methods (San Diego, Calif.) vol. 69, 1 (2014): 67-75.).

[0064] ③ The recovered products of steps (1) and (2) were connected with T4 ligase at 16℃ for 2 hours. In order to obtain better connection effect, it is recommended to place in 4℃ environment for several days.

[0065] ④ Transform DH5α competent E. coli

[0066] a. The frozen DH5α competent E. coli (TransGen, CD201-01) was taken out from -80℃ and thawed on ice. At this time, it is not recommended to use hand temperature to melt the E. coli. After fully dissolving, 5ul of the connection product obtained in step ③ was added to the DH5α competent E. coli solution, and it was mixed and placed on ice for several minutes.

[0067] b.42°C heat shock for 90 seconds, immediately placed on ice for 10 minutes, the meaning of this step is that after the time of incubation on ice, the DH5a competent E. coli transformed with the ligation plasmid will express ampicillin resistance protein, and if the heat shock is immediately cultured in LB liquid medium with ampicillin resistance, part of the DH5a competent E. coli which is not enough to express the resistance protein will be killed.

[0068] c. Add 250ul ampicillin (80ug / ml) resistant LB liquid medium, 37°C biochemical incubator 200rpm shaking incubation for 1 hour.

[0069] d. In the 4°C centrifuge, 4000rpm centrifugation for 1 minute, discard the supernatant, add 100ul LB liquid medium, resuspended after light, all the bacterial liquid was plated, 37°C inverted culture for 16 hours.

[0070] ⑤Cloning PCR screening positive clones

[0071] PCR primers (M13-47: 5'-cgccagggttttcccagtcacgac-3'; RV-M: 5'-agcggataacaatttcacacagga-3') were used to amplify the above-mentioned transformed bacterial liquid.

[0072] The length of the single-connector fragment is 102bp, the length of the primer amplified on the pT vector is 156bp, the length of the double-connector PCR product is 102x2+156=360bp, and the length of the n-connector PCR product should be (102xn+156)bp, and so on. Repeat the above method 2-3, insert pC and pG(NN) into the PMD18-T vector (containing pA, pT single-connector) which has been constructed, and finally obtain the required TALEN plasmid containing left and right arm RVDs sequence. Each time a positive clone is selected, LB liquid is shaken to expand the culture, and the plasmid is extracted with the kit, and the next step is repeated. Among them, the pC and pG(NN) vectors are obtained from the reference (Huang, Peng et al. "TALEN construction via "Unit Assembly" method and targeted genome modifications in zebrafish." Methods (San Diego, Calif.) vol. 69, 1 (2014): 67-75.).

[0073] 3.1.2 In vitro synthesis of target TALEN mRNA

[0074] 1) Plasmid linearization

[0075] The TALEN plasmid containing the left and right arm RVD sequences synthesized and screened in 3.1.1 above was single-enzymatically cut with NotI, and after 37°C overnight, loading buffer (TransGen, GH101-01) was added, mixed at a ratio of 1-fold loading buffer to 5-fold single-enzymatically cut product, and electrophoresed using 2% agarose gel, observed under ultraviolet light, and the plasmid that was not cut open was removed using a scalpel, and the agarose gel block containing the desired fragment was collected. Then the fragment in the agarose gel block was purified using a gel extraction kit, and finally the concentration was determined using a microspectrophotometer.

[0076] 2) In vitro synthesis of TALEN mRNA

[0077] The Ambion mMESSAGE mMACHINE (T7) kit was used for synthesis, and the system is shown in Table 1:

[0078] Table 1

[0079]

[0080] After 37°C incubation for 3 hours, 0.5 μl was taken for use. 1 ul TURBO DNase was added to the synthesis system to digest the template, mixed well, and then incubated at 37°C for 30 minutes. At this time, 0.5 μl of the sample before digestion was taken and run electrophoresis to determine whether the template was completely cut.

[0081] 3) Purification of mRNA

[0082] The TALEN target left arm mRNA chain and right arm mRNA chain were purified using Trizol. 500 μl Trizol was added to the TALEN mRNA (TALEN target left arm mRNA chain and right arm mRNA chain) synthesized in vitro in step 2) above, shaken vigorously for 15 s, and allowed to stand at room temperature for 5 min. Then, the organic layer was fully layered with the inorganic layer by centrifuging at 4°C for 10 minutes at maximum speed. The upper layer was then taken into an equal amount (equal volume) of isopropanol to precipitate the mRNA. After shaking well, the mixture was allowed to stand at room temperature for 10 minutes. Then, the mixture was centrifuged at 4°C for 10 minutes at maximum speed. At this time, white precipitate was observed at the bottom of the EP tube, which was the purified mRNA. The isopropanol was discarded, and 1 ml of 80% (v / v) ethanol solution was added to wash the mRNA. After shaking, the mixture was centrifuged at 7500g for 5 minutes, and the ethanol was removed and allowed to dry completely. The appropriate amount of DEPC water was added to dissolve the mRNA (TALEN target left arm mRNA chain and right arm mRNA chain), and the concentration was determined using a spectrophotometer. The mRNA was stored at -80°C for future use.

[0083] 4) Embryo microinjection of TALEN target mRNA

[0084] Mix the left arm mRNA strand and right arm mRNA strand of TALEN target site obtained after step 3 together, so that the final concentration is mRNA (100-200 ng / L), and take 2 uL sample into the injection needle tube. Adjust the air pressure so that the liquid volume of each injection is about 0.5 uL (droplet diameter is about 100 um). Collect the newly born AB wild type zebrafish embryos, and perform microinjection at the 1-2 cell stage, with the microinjection needle penetrating the yolk sac into the cells.

[0085] 5) Screening of zrb1-KO mutants

[0086] Lysate the embryos at 24 hpf, extract genomic DNA, and use PCR amplification to target the fragments near the site, using the primer sequences: FP: 5'-GCCACTGCTAAACACTAAAGA-3'; RP: 5'-GCTCCATGCCAGCAATAAAA-3. The obtained PCR product is then used to detect whether there is a mutation (which can be cut by T7endonuclease I, indicating that a mutation has occurred) using T7endonuclease I (New England Biolabs, M0302S), and the detection-efficient remaining embryos are cultured to grow up for subsequent F0 screening. After screening, the F0 is crossed with the AB wild type zebrafish to obtain the rb1 heterozygous mutant carrying the mutation, and two mutants are obtained by sequencing screening (2 base deletions in exon 2 of the rb1 gene: exon2:67-68; 8 base deletions in exon 2 of the rb1 gene: exon2:67-75), which are the F1 generation zrb1-KO + / - heterozygotes. The rb1 mutant with 2 base deletions in exon 2 is selected for subsequent experiments.

[0087] 4. Zebrafish behavioral analysis

[0088] The sibling AB wild type zebrafish (wt) and F1 generation zrb1-KO + / - heterozygote embryos are cultured to 5 dpf and then placed in a 48-well plate and detected by a zebrafish behavioral trajectory tracking system (DanioVision, Noldus), with reference to the specific literature (Colwill RM, Creton R. Imaging escape and avoidance behavior in zebrafish larvae. Rev Neurosci. 2011; 22(1): 63-73.) for detection. For the T maze, adult wt and zrb1-KO + / -The heterozygotes were trained for 4 days. Food was placed in the right arm to form an enrichment chamber (EC). After the training, the food was removed from the right arm and the residence time of the zebrafish in the left and right arms was measured. The experiment was set up in triplicate.

[0089] It was found that zrb1-KO mutant larvae exhibited abnormal motor function Figure 2 B and 2B'), zrb1-KO + / - The heterozygotes exhibited memory cognitive dysfunction Figure 2 C and 2C').

[0090] 5. AO staining

[0091] The principle of AO detecting apoptosis: Acridine Orange is a fluorescent dye that can bind to DNA double strands and insert between double strands, while DNA single strand or RNA is combined by electrostatic attraction. Under blue light excitation, the nucleus presents bright green fluorescence, and the RNA in the nucleolus and cytoplasm presents bright red fluorescence. The cation of acridine orange can also combine with protein and polysaccharide to emit fluorescence, but fixed cells can inhibit this combination, so that mainly two kinds of nucleic acids of DNA and RNA are shown.

[0092] The AO used in the application is configured as follows: 0.0500g of AO powder is accurately weighed using a one-in-ten thousand analytical balance, added to a centrifuge tube containing 10mL PTU (phenylthiourea, 30mg / L) in 15mL zebrafish embryo culture solution (the centrifuge tube is wrapped with tin foil to avoid light), the final concentration of AO is 5mg / mL, and it is dissolved in a shaking bed. After the powder is completely dissolved, it is stored in a 4℃ refrigerator in the dark, and before use, it is diluted to 5μg / mL with PTU zebrafish embryo culture solution at a dilution ratio of 1:1000.

[0093] Specific experimental steps:

[0094] 1) Soak the zebrafish at the appropriate developmental stage in the AO working solution (5μg / mL) for 1h.

[0095] 2) Remove the AO staining solution and wash with fish system water + PTU (30mg / L) 6 times, 5min each time (note that the AO staining solution and the washing solution, the gun head, etc. need to be collected specially and labeled as carcinogenic to prevent contamination).

[0096] 3) When the signal washing is visible, the zebrafish is anesthetized with 0.2% tricaine and fixed with 1% low-melting-point agarose.

[0097] 4) Laser confocal photography.

[0098] By confocal brightfield imaging, we observed a significant increase of apoptotic vesicles in the hindbrain of zrb1-KO mutants Figure 3 A and 3A’). Microglia cells, the innate immune cells of the brain, can concentrate in the area of neuronal death to eliminate apoptotic cellular debris. Therefore, we labeled microglia cells with neutral red (NR) staining and observed the expansion and infiltration of microglia cells in the cerebellum and medulla of zrb1-KO mutants Figure 3 B and 3B’). Consistent with these findings, AO staining (3 dpf) showed an increase of neuronal apoptosis in the cerebellum and medulla of zrb1-KO mutants Figure 3 C and 3C’). To further verify whether the induction of neuronal apoptosis in the hindbrain by rb1 deletion is an autonomous effect, we injected wild-type zrb1 mRNA into zrb1-KO embryos. The results showed that the injection of zrb1 mRNA in zrb1-KO mutants can partially rescue the phenotype of increased neuronal apoptosis in the cerebellum and medulla Figure 3 D and 3D’).

[0099] 6.10x single-cell transcriptome sequencing

[0100] 1) 10x Genomics Technology Overview

[0101] First, specific DNA fragments are seeded on the gel beads, which are composed of three parts: Barcode, UMI and PolyT. Barcode is 16 bases long. There are 4 million Barcodes in total, and one bead corresponds to one Barcode. Through these 4 million Barcodes, the gel beads can be distinguished. UMI is a random sequence, that is, each DNA molecule has its own UMI sequence. The 10-base-long UMI has 1 million sequence variations (4^10 = 1,048,576). The role of UMI is to distinguish which reads are from an original cDNA molecule, to distinguish between gene fragment repeats and duplications, and to distinguish between real SNP sites and PCR-induced mutations. Through the 10x Genomics instrument, single cells are mixed with single gel beads through an oil phase to form water-in-oil microdroplets. Next, the cell membrane is broken, and the mRNA in the cell is released. The released mRNA is mixed with the water phase in the microdroplet, that is, in contact with reverse transcriptase, nucleic acid primers combined with gel beads, and dNTP substrates. Next, the reverse transcription reaction is initiated. The mRNA is combined with the tagged DNA molecules on the gel beads, and under the action of reverse transcriptase, cDNA is reverse transcribed. All the water phases in the emulsion are extracted, that is, all the tagged cDNA molecules are extracted, and then the cDNA molecules are added with adaptors, amplified by PCR, made into Illumina sequencing libraries, and placed on the Illumina sequencer for sequencing. After sequencing is completed, data analysis is performed. The 10x Genomics technology can obtain multiple single-cell data at the same time, but only mRNA information can be obtained, and most LncRNA information is lost. The UMI technology can well remove the SNP sites introduced by duplication and PCR. The quality of RNA is also very high, and the degradation of RNA will also cause the loss of 5' end information.

[0102] 2) Preparation of brain single-cell suspension and sequencing analysis

[0103] a) After the siblings of AB wild-type zebrafish (wt) and zrb1-KO mutant zebrafish developed to 3 dpf were anesthetized, they were placed in a culture dish, the head and trunk were separated with a syringe, the head was placed in a clean 1.5 mL EP tube, at least 10 zebrafish in each sample, the residual liquid in the EP tube was sucked dry with a pipette, 200 μL of papain digestion solution (WBC, LS00 312) was added to the EP tube, and then the EP tube was placed in a 37°C metal bath for heating for 4 min.

[0104] b) Use a pipette to gently blow up the zebrafish head tissue block, then place the EP tube in a 37°C metal bath and heat for 2 minutes.

[0105] c) Continue pipetting the tissue sample 10 times to dissolve the tissue chunks into a suspension. Heat the sample in a 37°C metal bath for 2 minutes.

[0106] d) Pipette the tissue sample 5 times and then heat it in a 37°C metal bath for 2 minutes.

[0107] e) The tissue sample was gently pipetted 5 times again, and then placed in a 37°C metal bath to heat for 5 minutes.

[0108] f) Add 800 μL of wash buffer to each tube of sample to terminate papain digestion, and then centrifuge at 400g in a 4°C cold centrifuge for 5 minutes.

[0109] g) After discarding the supernatant, add 800 μL of wash buffer to each tube. Gently pipette to dissolve the tissue pellet. Centrifuge at 800 g for 5 minutes at 4°C.

[0110] h) Discard the supernatant and add 300 μL of washing buffer to dissolve the sample to obtain a brain single cell suspension sample.

[0111] i) Count and assay the cell viability of the brain single cell suspension. Once the cell suspension passes the assay, dilute it to an appropriate concentration and load it onto a 10x marker.

[0112] j) After labeling, the sample is reverse transcribed.

[0113] k) The protein was subsequently purified, library constructed, and sequenced by Kidio Biotechnology.

[0114] l) After the basic analysis results come out, we use 0.5 resolution to perform cell clustering, and then define cell groups by different characteristic genes. Next, we select the neural cell groups required for our experiment for cell re-clustering, re-definition and differential gene analysis ( Figure 4 A). The differentially expressed genes between the post-mitotic neuronal cell groups in the hindbrain (medulla oblongata neuronal cell group and cerebellar cell group) were analyzed through KEGG functional annotation. It was found that the differentially expressed genes in the medulla oblongata neuronal cell group and cerebellar cell group were enriched in the apoptosis signaling pathway ( Figure 4 B and 4C).

[0115] In addition, we used XhoI and AgeI to double-digest the Cas9-T2A-mCherry,U6:gRNA plasmid and inserted the nestin and huc promoters obtained by PCR into the Ultra One Step Cloning Kit (Vazyme, C115) was used to connect the enzyme-digested fragment of Cas9-T2A-mCherry, U6: gRNA plasmid to obtain two plasmids: nestin:Cas9-T2A-mCherry, U6: gRNA (rb1) and huc:Cas9-T2Am-Cherry, U6: gRNA (rb1). Since nestin and huc are expressed in NSPCs and postmitotic neurons, respectively, the two plasmids nestin:Cas9-T2A-mCherry, U6: gRNA (rb1) and huc:Cas9-T2Am-Cherry, U6: gRNA (rb1) can specifically knock out rb1 in NSPCs and postmitotic neurons. The results showed that inhibiting the expression of rb1 in postmitotic neurons induced apoptosis in the cerebellum and myelin ( Figure 4 D and 4D'), while inhibition of rb1 expression in NSPCs failed to induce cell apoptosis ( Figure 4 E and 4E'). Among them, the plasmid Cas9-T2A-mCherry, U6: gRNA reference (Luo, J. et al. Stepwise crosstalk between aberrant Nf1, Tp53 and Rb signalling pathways induces gliomagenesis in zebrafish. Brain: a journal of neurology 144, 615-635 (2021)) was obtained; the nestin promoter reference (Lam, CS, M.& U. gfap and nestin reporter lines reveal characteristics of neural progenitors in the adult zebrafish brain. Developmental dynamics : an official publication of the American Association of Anatomists 238, 475-486 (2009)) obtained; the huc promoter reference (Park, H. C. et al. Analysis of upstream elements in the HuC promoter leads to the establishment of transgenic zebrafish with fluorescent neurons. Dev Biol 227, 279-293 (2000)) obtained.

[0116] 7. Plasmid preparation

[0117] 1) Amplification of the fragment of interest

[0118] Taking the construction of the huc:kmt5b-egfp plasmid as an example. According to the position of the enzyme cutting sites on the pTol2 vector pTol2-DRv7 (Addgene, 78627) plasmid, we selected the PspOMI and XmaI double enzyme cutting sites as the insertion position of the kmt5b cDNA and Huc promoter fragment, and added homologous arm sequences and enzyme cutting site sequences to both the upstream primer and the downstream primer. The final synthesized kmt5b-FP, kmt5b-RP, Huc-FP and Huc-RP primer sequences are as follows:

[0119] kmt5b-FP: 5'-TCATCTTTGTACGTCAAGAATGGGAGAATCCAAGAACATGGTG-3';

[0120] kmt5b-RP: 5'-gctcaccatGGATCCCCCGGgTGCGTTGAGTCTGAGCGACTG-3';

[0121] Huc-FP: 5'-cttattttttggagatcacttGGGCCCGAATTCACTAATTTGAATTTAAATGCATTATCTTTCT-3';

[0122] Huc-RP: 5'-TTCTCCCATTCTTGACGTACAAAGATGATAGTGATCTAGGT-3'.

[0123] PCR reaction was performed using high-fidelity KOD polymerase, with cDNA of wild-type zebrafish at 3 dpf as template, to amplify kmt5b cDNA and Huc promoter fragment.

[0124] 2) Enzymatic digestion of the vector:

[0125] The position of the enzyme digestion site on the Ptol2 vector plasmid was selected for double digestion with PspOMI and XmaI. The enzyme digestion system was incubated at 37°C overnight. The next day, 5 μL of the enzyme digestion product and 1 uL of the original plasmid were mixed with ddH2O and 6x loading buffer to form a 6 μL system, and 1% agarose gel was used to analyze whether the enzyme digestion was complete. If the enzyme digestion was complete, 6x loading buffer was added to the remaining system, and 1% agarose gel electrophoresis was used to separate the gel. The agarose gel containing the completely cut fragments was cut under a UV lamp, and the gel was purified by GeneJET GEL extraction Kit to obtain a target fragment of about 10 kb in size.

[0126] 3) Homologous recombination: The purified and recovered products in steps 1) and 2) were subjected to homologous recombination at 50°C for 15 min using ULtra One Step Cloning Kit. The ligation product could be stored at 4°C for 7 days. The ligation system was as follows:

[0127] Table 2

[0128] Enzymatic linearization of the vector x μL Insert y μL 2x ClonExpress Mix 5 μL ddH2O up to 10 μL

[0129] In the table: 1. Optimal amount of cloning vector used (x) = [0.02 x number of base pairs of cloning vector] ng (0.03 pmol);

[0130] 2. Optimal amount of fragment used (y) = [0.02 x number of base pairs of each fragment] ng (0.03 pmol).

[0131] 4) Transformation of competent cells with the recombination product: (a) The chemical competent cells DH5a used for cloning were thawed on ice; (b) 5 μL of the recombination product was added to 50 μL of the competent cells, which were mixed gently and then incubated on ice for 15 min; (c) the cells were heat-shocked at 42°C for 90 s and then immediately cooled on ice for 10 min; (d) a sterile rod was used to evenly spread the cells on a plate containing the correct resistance; (e) the plate was inverted and incubated in a 37°C incubator for 12-16 h.

[0132] ​5) Colony PCR verification: pick several clones from the plate to do colony PCR to identify whether the colonies contain the correct colonies.

[0133] 6) Electrophoresis and sequencing verification: after PCR, add 1 μL loading buffer to 5 μL sample for agarose gel electrophoresis, and use an agarose gel electrophoresis imaging system to detect amplification. Select several colonies that amplify the correct band size for large-scale culture, extract the plasmid, and send it to a sequencing company for sequencing. After the sequencing results are obtained, select the correct plasmid for sequencing and name it huc:kmt5b-egfp plasmid, and store it in a -20°C refrigerator.

[0134] 7) Tunel staining

[0135] zrb1-KO + / - After the embryos produced by the self-crossing of heterozygotes, the above-constructed huc:kmt5b-egfp plasmid and ddH2O (control group) were injected into the embryos at the single-cell stage, and TUNEL staining was performed at 3 dpf to observe the effect on apoptosis. The principle of TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) detection of apoptosis: when apoptosis occurs, genomic DNA breaks, exposing 3'-OH which can be labeled with fluorescein-dUTP (fluorescein-dUTP) under the catalysis of terminal deoxynucleotidyl transferase (Terminal Deoxynucleotidyl Transferase), so that the situation of cell apoptosis can be understood through fluorescence microscopy or laser confocal microscopy. The Tunel experimental steps are as follows:

[0136] a) Fixation and dehydration: collect 4-day-old zebrafish embryos after fertilization in EP tubes, about 40-60 embryos. First, rinse the embryos with PBST for 2 times, 10 min each time, then add 4% paraformaldehyde 25°C room temperature to fix the embryos for 2h or 4°C overnight. Then, dehydrate the embryos with 50% methanol solution (50% methanol + 50% PBST, both by volume) and 100% methanol at room temperature, 5 min each time. Finally, replace 100% methanol and store at -20°C overnight or for long-term storage.

[0137] b) Rehydration: rehydrate with 75% methanol solution (75% methanol + 25% PBST), 50% methanol solution (50% methanol + 50% PBST) and 25% methanol solution (25% methanol + 75% PBST), 10 min each time, then wash the embryos with PBST 3 times, 5 min each time.

[0138] c) Digestion and permeabilization: 20 mg / ml of proteinase K was diluted with PBST at 1:2000 (volume ratio) and added to the embryos for digestion at 25 °C for 135 min, with the digestion time adjusted according to the specific room temperature if necessary, during which the embryos were shaken on a shaker at the minimum speed. The embryos were gently rinsed with PBST for 3 times, 10 min each time. The ethanol:acetone mixture was prepared and changed to a volume ratio of 2:1, added to the EP tube, and permeabilized at -20 °C for 7 min. The embryos were washed with PBST for 3 times, 10 min each time.

[0139] Results Figure 5 As shown, overexpression of bcl2a or inhibition of caspase in zrb1-KO mutants can reduce the number of apoptotic cells in the cerebellum and hindbrain Figure 5 A, Figure 5 A’, Figure 5 B and Figure 5 B’). Then, we analyzed the expression of known Rb1 binding proteins (such as E2Fs, Hdac1, Kmt5b, Dnmt1, etc.) using scRNA-seq data. The results showed that only Kmt5b (methyltransferase 5B, an enhancer of Rb1 binding proteins) was highly expressed in post-mitotic cerebellum and hindbrain neurons Figure 5 C). Therefore, overexpression of kmt5b in zrb1-KO mutants can partially rescue the apoptosis of post-mitotic neurons in the hindbrain Figure 5 D and Figure 5 D’), while knocking down kmt5b can partially mimic the phenotype of increased neuronal apoptosis in zrb1-KO mutants Figure 5 E and Figure 5 E’).

[0140] 8. R621S and L819V mutations of RB1 protein are associated with neuronal apoptosis

[0141] 1) hRB1 mRNA, hRB1 R621S mRNA and hRB1 L819V mRNA synthesis method refers to the above 3.1.2 in vitro synthesis of target TALEN mRNA, wherein the PCR primer sequences used are as follows:

[0142] hRB1-FP: 5'-aggatcccatcgattcgaattcatgccgcccaaaaccc-3';

[0143] hRB1-RP: 5'-ctcactatagttctagaGGCTCGAGctatttctcttccttgtttgaggtatccatgc-3';

[0144] 621FP: 5'-TTCAACTACG a GTGTAAATTCTACTGCAAATG-3';

[0145] 621RP: 5'-TTTACACt CGTAGTTGAACCTTTTTTCTTTG-3';

[0146] 819FP: 5'-GTGTGCCAACACCAACAAAAATGACT-3';

[0147] 819RP: 5'-GTGTTGGCACACCTTCTGAAATTTTATATGG-3'.

[0148] We verified the effects of human RB1 protein R621S and L819V changes on neuron apoptosis.

[0149] 2) The experiment was divided into 8 groups, and hRB1 mRNA, hRB1 R621S mRNA and hRB1 L819V mRNA were microinjected in sibling AB wild-type zebrafish and zrb1-KO mutants (the method is the same as 3.1.2 above), and the same volume of ddH2O was injected as a control. The experiment was set up three times.

[0150] The results show that injection of hRB1 mRNA in zrb1-KO mutants can partially rescue the cerebellum and brainstem neuron apoptosis phenotype Figure 6 A), but hRB1 R621S mRNA and hRB1 L819V mRNA do not change the number of apoptotic cells in the cerebellum and brainstem of zrb1-KO mutants Figure 6 A'). It is suggested that R621S and L819V mutations of RB1 protein may play a role in neuron apoptosis and are related to neurodegenerative diseases.

[0151] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application are equivalent replacement methods and are included in the protection scope of the present application.

Claims

1. Use of a mutant zebrafish in preparing an animal model for a neurodegenerative disease, characterized in that: The mutant zebrafish is a zrb1-KO mutant zebrafish; The zrb1-KO mutant zebrafish is a mutant formed by deleting two bases at positions 67 and 68 on exon 2 of the zebrafish rb1 gene.

2. The use according to claim 1, characterized in that: The zrb1-KO mutant zebrafish larvae exhibit abnormal motor function; Adult heterozygous zrb1-KO mutant zebrafish exhibit memory and cognitive dysfunction.

3. The use according to claim 1, characterized in that: The zrb1-KO mutant zebrafish has increased apoptosis of hindbrain neurons, which in turn causes an increase in hindbrain microglia.

4. The use according to claim 3, characterized in that: The apoptosis of hindbrain neurons in the zrb1-KO mutant zebrafish is postmitotic.

5. The use according to claim 3, characterized in that: The phenotype of increased apoptosis of hindbrain neurons in the zrb1-KO mutant zebrafish can be partially rescued by the mRNA of the zebrafish rb1 gene.

6. The use according to claim 1, characterized in that: The zrb1-KO mutant zebrafish is subjected to targeted gene knockout using TALEN technology; The TALEN target sequence of the zebrafish rb1 gene is as follows: The recognition sequence for the left arm is: CCAGTCCACTAACT; The right arm recognition sequence is: CTCCCATATTCTCCAT.

7. Use of an RB1 protein mutant in the preparation of a drug for diagnosing and / or treating a neurodegenerative disease, characterized in that: The RB1 protein mutant is at least one of R621S and L819V.

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