Application of Fc [gamma] RIIB as mammary gland IgG transporter

Abstract

The invention discloses an application of Fc [gamma] RIIB as a mammary gland IgG transporter of a mammal, and belongs to the technical field of biology. By constructing an Fc [gamma] RIIB knockout mouse and an Fc [gamma] RIIB knockout pig, the serum of the Fc [gamma] RIIB knockout mouse is found to be remarkably increased in milk IgG concentration ratio, namely, the relative concentration of milk IgG is reduced; although the content of IgG in serum of the Fc [gamma] RIIB-knocked-out pig is remarkably increased, IgG in colostrum is almost not detected. Therefore, it is proved for the first time that Fc [gamma] RIIB is a receptor for mediating transmammary transfer of serum IgG to milk, and a long-term unsolved problem in the field of maternal passive immunity research is solved. In addition, the invention provides a new strategy for improving the IgG content of colostrum and further improving the early immunity and survival rate of newborn livestock.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to the application of FcγRIIB as a receptor for transporting IgG in the mammary gland of mammals. Background Art

[0002] Maternal IgG is crucial for the better growth of offspring. Offspring can obtain maternal IgG through the following two pathways: one is through placental transfer before birth, directly entering the fetal blood circulation; the other is through sucking milk after birth. Maternal IgG reaches the milk from the serum, and after offspring suck the milk, it is absorbed in the small intestine. The process of crossing each cell layer barrier is recognized to be mediated by receptors. Among them, the placental cell layer barrier and the small intestinal cell layer barrier have been identified to be mediated by FcRn (Neonatal Fc Receptor). However, what the receptor is that mediates the crossing of the mammary gland cell layer barrier by IgG remains unclear. The prior art has identified that the transport receptors for the placental cell layer barrier and the small intestinal cell layer barrier for the passive transport of maternal IgG are FcRn, and FcRn is not the receptor for the transport of maternal IgG across the mammary gland cell layer barrier. Therefore, the identification of the passive transport receptor for maternal IgG is not perfect at present. Transmammary transport is the only way for large animal offspring to obtain maternal IgG. Therefore, the identification of the transport receptor for this barrier is of great significance. Summary of the Invention

[0003] In order to solve the above-mentioned deficiencies existing in the prior art, the present invention provides the application of FcγRIIB as a receptor for transporting IgG in the mammary gland of mammals, so as to increase the content of IgG in the milk of mammals.

[0004] The technical solution for the present invention to solve the above technical problems is as follows: Provide the application of an FcγRIIB gene or its encoded protein as a receptor for transporting IgG in the mammary gland of mammals. The FcγRIIB gene or its encoded protein mediates the transport of mammalian serum IgG across the mammary gland into the milk as a receptor.

[0005] Further, the mammals include mice, rats, pigs, cows, and sheep.

[0006] Further, the nucleotide sequence of mouse FcγRIIB is as shown in SEQ ID NO.1; the nucleotide sequence of rat FcγRIIB is as shown in SEQ ID NO.2; the nucleotide sequence of pig FcγRIIB is as shown in SEQ ID NO.3; the nucleotide sequence of cow FcγRIIB is as shown in SEQ ID NO.4; the nucleotide sequence of sheep FcγRIIB is as shown in SEQ ID NO.5; The amino acid sequence of the encoded protein of murine FcγRIIB is shown in SEQ ID NO.6; the amino acid sequence of the encoded protein of rat FcγRIIB is shown in SEQ ID NO.7; the amino acid sequence of the encoded protein of porcine FcγRIIB is shown in SEQ ID NO.8; the amino acid sequence of the encoded protein of bovine FcγRIIB is shown in SEQ ID NO.9; the amino acid sequence of the encoded protein of ovine FcγRIIB is shown in SEQ ID NO.10.

[0007] Furthermore, the IgG is at least one of murine 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 porcine IgG subtypes IgG1, IgG, IgG3, IgG4, and IgG5; the IgG is at least one of bovine IgG subtypes IgG1 and IgG2; the IgG is at least one of ovine IgG subtypes IgG1, IgG2, and IgG3.

[0008] The present invention provides a method for increasing the IgG content in mammalian milk, which overexpresses FcγRIIB in mammary tissue to increase the IgG content in mammalian milk.

[0009] Furthermore, overexpressing FcγRIIB includes the following steps: constructing a mammary gland-specific overexpression vector of FcγRIIB, and then performing microinjection on the overexpression vector.

[0010] Furthermore, the backbone of the overexpression vector is the pBC1 expression vector.

[0011] The present invention has the following beneficial effects: By constructing FcγRIIB knockout mice and FcγRIIB knockout pigs, it is found that the serum and milk IgG ratios of FcγRIIB knockout mice are significantly increased, that is, the relative concentration of milk IgG is decreased; although the serum IgG content of FcγRIIB knockout pigs is significantly increased, almost no IgG can be detected in the colostrum. Therefore, the present invention first proves that FcγRIIB is the receptor that mediates the trans-mammary transport of serum IgG into milk, answering a long-standing unresolved question in the field of maternal passive immunity research. By specifically overexpressing FcγRIIB in murine mammary tissue and obtaining FcγRIIB overexpressing mice, it is found that the milk IgG content of these mice is significantly increased while the serum IgG remains almost unchanged, that is, there is an enrichment phenomenon of milk IgG in the overexpressing mice. The present invention provides a new strategy for improving the early immunity and survival rate of neonatal livestock. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 For Fcgr1 andFcgr4 Knockout mouse construction strategy; Figure 2 For Fcgr2b Detection of IgG transport in the milk of knockout mice; Figure 3 For Fcgr2b Detection of IgG transport in the milk of conditional knockout mice; Figure 4 For the expression of FcγRIIB in porcine perinatal mammary tissue; Figure 5 For the map of PX458 vector; Figure 6 For the construction of FcγRIIB knockout pig model; Figure 7 For the detection of IgG transport in the milk of FcγRIIB knockout pigs; Figure 8 For the map of pBC1-FcγRIIB1 / FcγRIIB2 expression vector; Figure 9 For the qPCR detection of FcγRIIB in the mammary tissue of transgenic mice; Figure 10 For the detection of IgG transport in the milk of FcγRIIB overexpressing mice. Detailed implementation methods

[0013] The following examples are only used to explain the present invention and are not intended to limit the scope of the present invention. For those not specified in the examples, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be purchased commercially.

[0014] Example 1: Preparation of FcγRs knockout mice and detection of IgG transport in milk In mice, there are three other receptors belonging to the same family as FcγRIIB, namely FcγI, FcγRIII, and FcγRIV. Therefore, mouse models with the four receptor genes knocked out respectively (see Figure 1 ) were used to identify the mammary IgG transport receptor. FcγRIIB knockout mice and FcγRIII knockout mice were purchased from Jackson Lab. The construction methods of FcγRI knockout mice and FcγRIV knockout mice are as follows: According to the mouse Fcgr1 , Fcgr4 gene sequences, 4 gRNAs were designed for each, and 2 gRNAs were designed at each target position to be cut. The gRNAs with higher off-target scores were preferentially used. Amplify Fcgr4- The DNA fragment of sgRNA was then recovered by gel extraction to serve as the template for in vitro transcription of sgRNA, followed by in vitro transcription and purification of sgRNA. The purified sgRNA and Cas9-mRNA were co-injected into C57 mouse embryos, and after injection, the embryos were transplanted into the oviducts of surrogate recipient mice until the mice were born. Among them, the nucleotide sequences of the 4 gRNAs are shown as follows: Fcgr1 - gRNA1: 5'-GATACTTGCATCGTATCCTT-3' (SEQ ID NO.11); Fcgr1 - gRNA2: 5'-CTGACACGCAGGCCGTCCCT-3' (SEQ ID NO.12); Fcgr4 - gRNA1: 5'-ATGGAACATGACTCTGTCGA-3' (SEQ ID NO.13); Fcgr4 - gRNA2: 5'-GAGCCGGTTGATAATATCTG-3' (SEQ ID NO.14).

[0015] The sandwich ELISA method was used to measure the IgG content in the postpartum serum and milk of the above 4 knockout mice, and the ratio analysis (Ratio-Serum / Milk) of the serum and milk IgG content was performed to measure the IgG transport in milk. The above method was used for measurement and analysis in the subsequent examples. The results showed that only the ratio of serum to milk IgG concentration in FcγRIIB knockout mice increased significantly, that is, the relative concentration of milk IgG decreased (see Figure 2 ), indicating that FcγRIIB may mediate the transport of serum IgG to milk in mice.

[0016] Example 2: Preparation of FcγRIIB conditional knockout mice and detection of milk IgG transport Since the change in serum IgG content in FcγRIIB knockout mice would affect the change in milk IgG content, a mouse model with specific knockout of FcγRIIB in the mammary gland was constructed using the Cre / Loxp system. MMTV-Cre and K14-Cre tool mice were respectively 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, and K14-Cre mice and FcγRIIB Flox mice were donated by other research groups).

[0017] The sandwich ELISA method was used to measure the IgG content in the postpartum serum and milk of FcγRIIB conditional knockout mice, and the ratio of serum IgG content to milk IgG content was analyzed to evaluate the IgG transport in milk. The results showed that there was no significant change in the serum IgG content of these mice, while the milk IgG concentration decreased significantly, and the ratio of the two increased significantly, indicating a decrease in the relative concentration of milk IgG (see Figure 3 ), suggesting that FcγRIIB mediates the transport of serum IgG to milk in mice.

[0018] Example 3: Construction of FcγRIIB knockout pigs and detection of colostrum IgG transport The expression of FcγRIIB in porcine perinatal mammary tissue was detected. The results showed that FcγRIIB was highly expressed in prenatal mammary tissue and its expression decreased after parturition (see Figure 4 ). Subsequently, FcγRIIB knockout pigs were prepared using CRISPR / Cas9 and somatic cell nuclear transfer technology. The construction method was to clone sgRNA into the PX458 vector (vector map see Figure 5 ), and then electrotransfect porcine ear fibroblasts with vigorous growth and good condition. The genomic DNA of the cells was extracted, primers were designed near the FCGR2B target site, and the genomic sequence containing the target site was amplified by high-fidelity enzyme PCR. TA cloning was performed, and about 20 single colonies were selected for PCR detection for each target site. Colonies with the correct band size were sent for sequencing, and the mutation types of the sequences were counted and the proportion of the number of mutant type sequences to the number of sequenced sequences was calculated, so as to screen out the 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)). Then, monoclonal cell lines were selected at these two sgRNA target sites. The recombinant PX458 plasmids ligated with these two gRNAs were electrotransfected into fetal ear fibroblasts of Wuzhishan boars and sows respectively. After culturing for 48 h, the cells were collected and made into cell suspensions, and flow cytometry sorting was performed using the GFP green fluorescence signal carried by PX458; the sorted cells were cultured by the limiting dilution method at a density of 150 - 200 cells per 10 cm culture dish. When the cells grew into monoclonal colonies (about 10 d), monoclonal cells with regular edges and vigorous growth were picked with a cell cloning ring into the culture dishes of 96-well plates. When the cell confluence reached more than 80%, the cells were successively passaged into 48-well, 24-well, 12-well and 6-well plate culture dishes. Some cells were collected for genomic DNA extraction and mutation type identification, and some cells were cryopreserved. After screening for targeting FCGR2BAfter obtaining monoclonal cell lines of sows and boars for gene exons 1 and 3, cells with bi-allelic knockout and the same base mutation types on both chromosomes were selected for somatic cell nuclear transfer. The donor cell variety for transplantation was Wuzhishan pigs, and finally cloned pigs were obtained. Western Blotting detected that FcγRIIB was successfully deleted in various tissues of the cloned pigs (see Figure 6 ).

[0019] The sandwich ELISA method was used to measure the IgG content in the postpartum serum and milk of FcγRIIB knockout sows, and the ratio of serum and milk IgG content was analyzed to measure the IgG transport in milk. The results showed that the IgG content in the serum of the knockout pigs increased significantly, while the IgG in milk was hardly detectable, and the ratio between the two increased extremely significantly, indicating that FcγRIIB is the receptor mediating the transport of porcine serum IgG to milk (see Figure 7 ).

[0020] Example 4: Preparation of FcγRIIB overexpressing mice and detection of IgG transport in milk The pBC1 expression vector can achieve specific overexpression of foreign genes in mammary tissues. Therefore, the pBC1-FcγRIIB1 / FcγRIIB2 expression vector was constructed by restriction enzyme digestion and ligation methods (see Figure 8 ). After double digestion of the expression vector with Sal I / Not I and linearization, microinjection was performed, and the transgenic F0 generation mice were successfully obtained. qPCR detected that FcγRIIB was successfully overexpressed in the mammary tissues of the transgenic mice (see Figure 9 ).

[0021] The sandwich ELISA method was used to measure the IgG content in the postpartum serum and milk of FcγRIIB overexpressing mice, and the ratio of serum and milk IgG content was analyzed to measure the IgG transport in milk. The results showed that the IgG content in the serum of FcγRIIB overexpressing mice remained almost unchanged, while the IgG content in milk increased significantly, and the ratio between the two decreased significantly, that is, the phenomenon of IgG enrichment in milk occurred (see Figure 10 ).

[0022] In summary, the present invention for the first time proves that FcγRIIB is the receptor mediating the transport of serum IgG across the mammary gland to milk, answering a long-standing unresolved problem in the field of maternal passive immunity research. Moreover, the present invention provides a new strategy for increasing the IgG content in colostrum and thus improving the early immunity and survival rate of neonatal livestock.

[0023] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. Use of FcγRIIB gene or its encoded protein as a mammalian mammary gland IgG transport receptor, characterized in that: The FcγRIIB gene or its encoded protein acts as a receptor to mediate the transport of mammalian serum IgG across the mammary gland into milk.

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

3. The use according to claim 2, characterized in that: The nucleotide sequence of the mouse FcγRIIB is shown in SEQ ID NO.1; the nucleotide sequence of the rat FcγRIIB is shown in SEQ ID NO.2; the nucleotide sequence of the pig FcγRIIB is shown in SEQ ID NO.3; the nucleotide sequence of the bovine FcγRIIB is shown in SEQ ID NO.4; the nucleotide sequence of the sheep FcγRIIB is shown in SEQ ID NO.5; The amino acid sequence of the protein encoded by the mouse FcγRIIB is shown in SEQ ID NO.6; the amino acid sequence of the protein encoded by the rat FcγRIIB is shown in SEQ ID NO.7; the amino acid sequence of the protein encoded by the pig FcγRIIB is shown in SEQ ID NO.8; the amino acid sequence of the protein encoded by the bovine FcγRIIB is shown in SEQ ID NO.9; the amino acid sequence of the protein encoded by the sheep FcγRIIB is shown in SEQ ID NO.

10.

4. The 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 bovine IgG subtypes IgG1 and IgG2; the IgG is at least one of sheep IgG subtypes IgG1, IgG2 and IgG3.

5. A method for increasing the IgG content in mammalian milk, characterized in that: Overexpression of FcγRIIB in mammary tissue increases the IgG content in mammalian milk.

6. The method according to claim 5, characterized in that The overexpression of FcγRIIB comprises the following steps: constructing an FcγRIIB mammary gland-specific overexpression vector, and then microinjecting the overexpression vector.

7. The method according to claim 6, characterized in that The skeleton of the overexpression vector is the pBC1 expression vector.

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