Use of fcγRIIB as mammalian mammary igg transport receptor

Abstract

The use of FcγRIIB as a mammalian mammary IgG transport receptor. By constructing FcγRIIB-knockout mice and FcγRIIB-knockout pigs, it is found that the ratio of serum and milk IgG concentrations of the FcγRIIB-knockout mice is significantly increased, that is, the relative concentration of milk IgG is reduced; and the FcγRIIB-knockout pigs have a significantly increased serum IgG content, but IgG is nearly undetectable in colostrum. Therefore, it has been proved for the first time that FcγRIIB is a receptor that mediates the trans-mammary transport of serum IgG to milk, and answers a long-term unsolved problem in the field of maternal passive immunity research. Furthermore, a new strategy is provided for increasing the IgG content of colostrum, thereby improving the early immunity and survival rate of neonatal domestic animals.

Description

Application of FcγRIIB as a mammalian mammary IgG transport receptor Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to the application of FcγRIIB as a mammalian mammary IgG transport receptor. Background Technology

[0002] Maternal IgG is crucial for the better growth of offspring. Offspring acquire maternal IgG through two pathways: first, prenatal transport via the placenta, directly entering the fetal bloodstream; and second, postnatal absorption through breast milk, where maternal IgG travels from the serum to the milk and is absorbed in the small intestine. The process of crossing various cell barriers is generally accepted to be mediated by receptors. The placental and small intestinal cell barriers have been identified as being mediated by the Neonatal Fc Receptor (FcRn). However, the receptor mediating IgG transport across the mammary cell barrier remains unclear. Current technology has identified FcRn as the transport receptor for passive maternal IgG transport across the placental and small intestinal cell barriers, but FcRn is not the receptor for mammary IgG transport across the mammary cell barrier. Therefore, the identification of passive maternal IgG transport receptors is currently incomplete. Cross-mammary transport is the only route for offspring in large animals to acquire maternal IgG; therefore, the identification of transport receptors for this barrier is of great significance. Summary of the Invention

[0003] To address the aforementioned shortcomings of the existing technology, this invention provides the application of FcγRIIB as a mammalian mammary IgG transport receptor to increase the IgG content in mammalian milk.

[0004] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: providing an application of the FcγRIIB gene or its encoded protein as a mammalian mammary IgG transport receptor, wherein the FcγRIIB gene or its encoded protein acts as a receptor to mediate the transport of mammalian serum IgG across the mammary gland to milk.

[0005] Furthermore, mammals include mice, rats, pigs, cattle, and sheep.

[0006] Furthermore, the nucleotide sequence of mouse FcγRIIB is shown in SEQ ID NO.1; the nucleotide sequence of rat FcγRIIB is shown in SEQ ID NO.2; the nucleotide sequence of pig FcγRIIB is shown in SEQ ID NO.3; the nucleotide sequence of bovine FcγRIIB is shown in SEQ ID NO.4; and the nucleotide sequence of sheep FcγRIIB is shown in SEQ ID NO.5.

[0007] The amino acid sequence of the protein encoded by mouse FcγRIIB is shown in SEQ ID NO.6; the amino acid sequence of the protein encoded by rat FcγRIIB is shown in SEQ ID NO.7; the amino acid sequence of the protein encoded by pig FcγRIIB is shown in SEQ ID NO.8; the amino acid sequence of the protein encoded by bovine FcγRIIB is shown in SEQ ID NO.9; and the amino acid sequence of the protein encoded by sheep FcγRIIB is shown in SEQ ID NO.10.

[0008] Furthermore, IgG is at least one of mouse IgG subtypes IgG1, IgG2a, IgG2b, IgG2c and IgG3; IgG is at least one of rat IgG subtypes IgG1, IgG2a, IgG2b and IgG2c; IgG is at least one of porcine IgG subtypes IgG1, IgG, IgG3, IgG4 and IgG5; IgG is at least one of bovine IgG subtypes IgG1 and IgG2; and IgG is at least one of sheep IgG subtypes IgG1, IgG2 and IgG3.

[0009] This invention provides a method for increasing the IgG content in mammalian milk by overexpressing FcγRIIB in mammary gland tissue to increase the IgG content in mammalian milk.

[0010] Furthermore, the overexpression of FcγRIIB includes the following steps: constructing a breast-specific overexpression vector for FcγRIIB, and then microinjecting the overexpression vector.

[0011] Furthermore, the backbone of the overexpression vector is the pBC1 expression vector.

[0012] This invention offers the following advantages: By constructing FcγRIIB knockout mice and FcγRIIB knockout pigs, it was found that the serum-to-milk IgG ratio was significantly increased in FcγRIIB knockout mice, indicating a decrease in the relative concentration of IgG in milk; while in FcγRIIB knockout pigs, although serum IgG levels were significantly increased, colostrum IgG was almost undetectable. Therefore, this invention demonstrates for the first time that FcγRIIB is a receptor mediating the transmammary transport of serum IgG to milk, answering a long-standing question in the field of maternal passive immunization research. Specific overexpression of FcγRIIB in mouse mammary tissue yielded FcγRIIB-overexpressing mice. These mice showed a significant increase in milk IgG levels while maintaining almost no change in serum IgG levels, indicating an enrichment of IgG in the milk of the overexpressing mice. This invention provides a new strategy for improving early immunity and survival rates in newborn livestock. Attached Figure Description

[0013] Figure 1 shows the strategy for constructing Fcgr1 and Fcgr4 knockout mice;

[0014] Figure 2 shows the detection of IgG transport in the milk of Fcgr2b knockout mice;

[0015] Figure 3 shows the detection of IgG transport in the milk of Fcgr2b conditional knockout mice;

[0016] Figure 4 shows the expression of FcγRIIB in porcine mammary gland tissue during the peripartum period;

[0017] Figure 5 shows the spectrum of the PX458 vector;

[0018] Figure 6 shows the construction of the FcγRIIB knockout pig model;

[0019] Figure 7 shows the detection of IgG transport in milk from FcγRIIB knockout pigs;

[0020] Figure 8 shows the pBC1-FcγRIIB1 / FcγRIIB2 expression vector map;

[0021] Figure 9 shows the qPCR detection results of FcγRIIB in mammary tissue of transgenic mice;

[0022] Figure 10 shows the detection of IgG transport in the milk of FcγRIIB overexpressing mice. Detailed Implementation

[0023] The examples given below are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, conditions in the examples are performed under standard conditions or as recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0024] Example 1: Preparation of FcγRs knockout mice and detection of IgG transport in breast milk

[0025] In mice, three other receptors belonging to the same family as FcγRIIB exist: FcγI, FcγRIII, and FcγRIV. Therefore, mouse models with knockout of each of the four receptor genes (see Figure 1) were used to identify mammary IgG transport receptors. FcγRIIB and FcγRIII knockout mice were purchased from Jackson Lab. The construction methods for FcγRI and FcγRIV knockout mice are as follows: Four gRNAs were designed based on the mouse Fcgr1 and Fcgr4 gene sequences, with two gRNAs designed for each target cleavage site, prioritizing gRNAs with higher off-target scores. The Fcgr4-sgRNA DNA fragment was amplified using sgRNA-pX330 as a template, then gel-recovered as a template for in vitro transcription of sgRNA. The sgRNA was then transcribed and purified. The purified sgRNA and Cas9-mRNA were co-injected into C57 mouse embryos, which were then transferred to the oviducts of surrogate recipient mice until birth. The nucleotide sequences of the four gRNAs are shown below:

[0026] Fcgr1-gRNA1: 5'-GATACTTGCATCGTATCCTT-3' (SEQ UID NO. 11);

[0027] Fcgr1-gRNA2: 5'-CTGACACGCAGGCCGTCCCT-3' (SEQ UID NO. 12);

[0028] Fcgr4-gRNA1: 5'-ATGGAACATGACTCTGTCGA-3' (SEQ UID NO. 13);

[0029] Fcgr4-gRNA2: 5'-GAGCCGGTTGATAATATCTG-3' (SEQ UID NO. 14).

[0030] The levels of serum and milk IgG in the four knockout mice were measured using a sandwich ELISA method. The ratio of serum to milk IgG levels was analyzed to measure milk IgG transport. Subsequent examples also used the same method for measurement and analysis. The results showed that only the FcγRIIB knockout mice exhibited a significantly increased serum-to-milk IgG concentration ratio, indicating a decreased relative milk IgG concentration (see Figure 2). This suggests that FcγRIIB may mediate the transport of mouse serum IgG to milk.

[0031] Example 2: Preparation of FcγRIIB conditional knockout mice and detection of IgG transport in breast milk

[0032] Since changes in serum IgG levels in FcγRIIB knockout mice affect changes in breast milk IgG levels, a breast-specific FcγRIIB knockout mouse model was constructed using the Cre / Loxp system. MMTV-Cre and K14-Cre mice were mated with FcγRIIB Flox mice to specifically delete Fcgr2b in mammary myoepithelial cells and luminal epithelial cells (MMTV-Cre mice were purchased from Cyagen Biosciences, while K14-Cre and FcγRIIB Flox mice were donated by other research groups).

[0033] The levels of serum and milk IgG in FcγRIIB conditional knockout mice were measured using a sandwich ELISA method. The ratio of serum to milk IgG levels was analyzed to assess milk IgG transport. The results showed no significant change in serum IgG levels, while milk IgG concentration was significantly reduced, and the ratio was significantly increased, indicating a decrease in the relative concentration of milk IgG (see Figure 3). This suggests that FcγRIIB mediates the transport of mouse serum IgG to milk.

[0034] Example 3: Construction of FcγRIIB knockout pigs and detection of colostrum IgG transport

[0035] The expression of FcγRIIB in porcine mammary tissue during the peripartum period was detected. The results showed that FcγRIIB was highly expressed in prepartum mammary tissue and decreased after parturition (see Figure 4). Subsequently, FcγRIIB knockout pigs were prepared using CRISPR / Cas9 and nuclear transfer technology. The construction method was to clone sgRNA into the PX458 vector (vector map shown in Figure 5) and electrotransfect it into vigorous and healthy porcine ear fibroblasts. Genomic DNA was extracted from cells, and primers were designed near the FCGR2B target site. High-fidelity PCR was used to amplify the genomic sequence containing the target site. TA cloning was performed, and approximately 20 single colonies were selected from each target site for PCR detection. Colonies with the correct band size were sent for testing. The mutation types of the sequences were counted, and the proportion of mutated sequences to the total number of sequences sent for testing was calculated. This screened out highly efficient targeting sgRNAs 1-3 and 3-3 (see Figure 6, where 1-3: 5'-ACAGGAGCATGTGGCCCAAAGG-3' (SEQ UID NO.15); 3-3: 5'-GGAGCACATTGATCCATGCAGG-3' (SEQ UID NO.16)). Single-clone cell lines were then selected from these two sgRNA target sites. Fetal ear fibroblasts from Wuzhishan boars and sows were electroporated with the two recombinant PX458 plasmids linked to gRNAs. After 48 hours of culture, cells were collected and prepared into a cell suspension. Flow cytometry was used to sort the cells using the GFP green fluorescence signal inherent in PX458. The sorted cells were then cultured using a limiting dilution method at a density of 150-200 cells per 10 cm culture dish. After the cells grew to monoclonal status (approximately 10 days), well-defined, vigorous monoclonal cells were picked using a cell cloning loop and transferred to 96-well plates. When the cell confluence reached over 80%, the cells were sequentially passaged into 48-well, 24-well, 12-well, and 6-well plates. Some cells were collected for genome extraction and mutation identification, while the remaining cells were cryopreserved. After obtaining monoclonal cell lines from sows and boars targeting the FCGR2B gene exon1 and exon3, cells with biallelic gene knockout and identical base mutation types on both chromosomes were selected for somatic cell nuclear transfer. All donor cells were from Wuzhishan, resulting in cloned pigs. Western blotting confirmed successful deletion of FcγRIIB in all tissues of the cloned pigs (see Figure 6).

[0036] The levels of IgG in the postpartum serum and milk of FcγRIIB knockout sows were measured using a sandwich ELISA method. The ratio of serum to milk IgG levels was analyzed to assess IgG transport in milk. The results showed that serum IgG levels were significantly elevated in knockout sows, while milk IgG was almost undetectable, and the ratio was significantly increased, indicating that FcγRIIB is the receptor mediating the transport of IgG from porcine serum to milk (see Figure 7).

[0037] Example 4: Preparation of FcγRIIB overexpression mice and detection of IgG transport in milk

[0038] The pBC1 expression vector can achieve specific overexpression of exogenous genes in mammary tissue. Therefore, the pBC1-FcγRIIB1 / FcγRIIB2 expression vector was constructed using enzyme digestion and ligation (see Figure 8). After linearization by double digestion with Sal I / Not I, the expression vector was microinjected, and F0 generation transgenic mice were successfully obtained. qPCR detection showed that FcγRIIB was successfully overexpressed in the mammary tissue of the transgenic mice (see Figure 9).

[0039] The levels of IgG in postpartum serum and milk of FcγRIIB-overexpressing mice were measured using a sandwich ELISA method. The ratio of serum to milk IgG levels was analyzed to assess milk IgG transport. The results showed that the serum IgG level of FcγRIIB-overexpressing mice remained almost unchanged, while the milk IgG level increased significantly, and the ratio of the two levels decreased significantly, indicating that milk IgG was enriched (see Figure 10).

[0040] In summary, this invention is the first to demonstrate that FcγRIIB is a receptor mediating the transmammary IgG transport to milk, answering a long-standing question in the field of maternal passive immunization research. Furthermore, this invention provides a new strategy for increasing colostrum IgG levels, thereby improving early immunity and survival rates in newborn livestock.

[0041] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Use of the FcγRIIB gene or the protein encoded thereby as an IgG transport receptor in the mammary gland of a mammal, characterized in that, The Fc gamma RII B gene or the protein encoded thereby is used as a receptor to mediate the transport of serum IgG in mammals across the mammary gland to milk.

2. Use according to claim 1, characterized in that, The mammals include mice, rats, pigs, cows and sheep.

3. Use according to claim 2, characterized in that, The nucleotide sequence of the mouse Fc gamma RII B is shown as SEQ ID NO. 1; the nucleotide sequence of the rat Fc gamma RII B is shown as SEQ ID NO. 2; the nucleotide sequence of the pig Fc gamma RII B is shown as SEQ ID NO. 3; the nucleotide sequence of the cow Fc gamma RII B is shown as SEQ ID NO. 4; and the nucleotide sequence of the sheep Fc gamma RII B is shown as SEQ ID NO.

5. The amino acid sequence of the protein encoded by the mouse Fc gamma RII B is shown as SEQ ID NO. 6; the amino acid sequence of the protein encoded by the rat Fc gamma RII B is shown as SEQ ID NO. 7; the amino acid sequence of the protein encoded by the pig Fc gamma RII B is shown as SEQ ID NO. 8; the amino acid sequence of the protein encoded by the cow Fc gamma RII B is shown as SEQ ID NO. 9; and the amino acid sequence of the protein encoded by the sheep Fc gamma RII B is shown as SEQ ID NO.

10.

4. Use according to claim 2, characterized in that, The IgG is at least one of mouse IgG subtypes IgG1, IgG2a, IgG2b, IgG2c and IgG3; the IgG is at least one of rat IgG subtypes IgG1, IgG2a, IgG2b and IgG2c; the IgG is at least one of pig IgG subtypes IgG1, IgG, IgG3, IgG4 and IgG5; the IgG is at least one of cow IgG subtypes IgG1 and IgG2; and the IgG is at least one of sheep IgG subtypes IgG1, IgG2 and IgG3.

5. A method of increasing the content of IgG in the milk of a mammal, characterized in that, The Fc gamma RII B is overexpressed in mammary tissue to increase the content of IgG in milk of mammals.

6. The method of claim 5, wherein, The overexpression of Fc gamma RII B includes the following steps: constructing a mammary gland-specific overexpression vector of Fc gamma RII B, and then performing microinjection on the overexpression vector.

7. The method of claim 6, wherein, The backbone of the overexpression vector is a pBC1 expression vector.

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