猪血型PCR鉴定方法的建立

doi:10.12300/j.issn.1674-5817.2023.065

实验动物与比较医学 ›› 2023, Vol. 43 ›› Issue (6): 585-594.DOI: 10.12300/j.issn.1674-5817.2023.065

• 动物实验技术与方法 • 下一篇

猪血型PCR鉴定方法的建立

王娇祥¹^,²^,³, 王艳¹^,²^,⁴, 胡珂¹^,²^,³, 徐凯祥¹^,²^,⁴, 魏太云¹^,², 角德灵¹^,²^,⁴, 赵恒¹^,²^,⁴, 赵红业¹^,²^,⁴, 魏红江¹^,²^,³^,⁴()()

^1.云南省小型猪基因编辑与异种器官移植重点实验室, 昆明 650201
^2.云南省异种器官移植工程研究中心, 昆明 650201
^3.云南农业大学动物科学技术学院, 昆明 650201
^4.云南农业大学动物医学院, 昆明 650201

收稿日期:2023-05-25 修回日期:2023-08-14 出版日期:2023-12-25 发布日期:2024-01-06
通讯作者: 魏红江（1971—），男，博士，教授，主要从事动物基因编辑与体细胞克隆技术研究。E-mail： hongjiangwei@126.com。ORCID： 0000-0002-5663-1093
作者简介:王娇祥（1996—），女，博士研究生，主要从事基因编辑研究。E-mail： jiaoxiangwang2021@126.com；共同第一作者
王艳（1997—），女，硕士研究生，主要从事基础兽医学研究。E-mail： 2788063601@qq.com；共同第一作者
基金资助:
国家重点研发计划干细胞及转化研究重点专项“异种移植用人源化基因编辑供体猪的构建及临床前研究”(2019YFA0110700);云南省重大科技专项计划“猪-非人灵长类异种器官移植关键技术的创新与应用”(202102AA310047);云南省创新团队“云南农业大学动物基因编辑和体细胞克隆技术省创新团队（带头人：魏红江）”(2017HC017)

Establishment of PCR Identification Method for Pig Blood Type

Jiaoxiang WANG¹^,²^,³, Yan WANG¹^,²^,⁴, Ke HU¹^,²^,³, Kaixiang XU¹^,²^,⁴, Taiyun WEI¹^,², Deling JIAO¹^,²^,⁴, Heng ZHAO¹^,²^,⁴, Hongye ZHAO¹^,²^,⁴, HongJiang WEI¹^,²^,³^,⁴()()

^1.Key Laboratory of Animal Gene Editing and Animal Cloning in Yunnan Province, Kunming 650201, China
^2.Xenotransplantation Engineering Research Center in Yunnan Province, Kunming 650201, China
^3.College of Animal Science and Technology, Yunnan Agricultural University, Kunming 650201, China
^4.College of Veterinary Medicine, Yunnan Agricultural University, Kunming 650201, China

Received:2023-05-25 Revised:2023-08-14 Published:2023-12-25 Online:2024-01-06
Contact: WEI HongJiang (ORCID: 0000-0002-5663-1093), E-mail: hongjiangwei@126.com

摘要/Abstract

摘要：

目的异种器官移植是解决人类器官供体短缺的有效途径，但其面临严重的免疫排斥反应，包括血型差异导致的超急性排斥反应。建立稳定、便捷、可靠的猪血型鉴定方法，可快速筛选合适血型的供体猪用于异种器官移植研究。方法选择版纳微型猪近交系、滇南小耳猪、巴马香猪为研究对象，用DNA提取试剂盒提取3种品系猪的血液、口腔颊黏膜和胎儿成纤维细胞中DNA，通过PCR法对猪ABO血型同源基因EAA第7号内含子附近的目标片段进行扩增。采用血液凝集反应检测猪前腔静脉全血加入抗A、B抗体后的溶血现象，采用免疫组织化学法检测猪心、肝、脾、肺、肾组织中A抗原表达水平，采用免疫荧光法检测猪口腔黏膜中A抗原表达水平。通过对比不同方法测定猪血型的结果，验证PCR方法的稳定性和可靠性，以此建立一种基于PCR的便捷式猪血型鉴定方法。结果首先，69头版纳微型猪近交系、7头滇南小耳猪和34头巴马香猪的血液PCR结果显示AO血型20头，AA血型66头，OO血型24头。47头滇南小耳猪的胎儿成纤维细胞PCR结果显示47个胎儿均为OO血型，其中8头基因编辑克隆猪的口腔黏膜PCR结果与供体胎儿成纤维细胞的PCR结果一致，均为OO血型；8头野生型猪（2头版纳微型猪近交系、4头滇南小耳猪和2头巴马香猪）的口腔黏膜PCR结果均与血液PCR鉴定结果一致。然后，从中随机挑选11头版纳微型猪近交系、4头滇南小耳猪和2头巴马香猪进行血液凝集反应验证，结果均与血液及口腔黏膜PCR鉴定结果一致。而且，对1头版纳微型猪近交系和2头巴马香猪的心、肝、肺、肾、脾组织进行免疫组织化学分析，结果与血液PCR鉴定和血液凝集反应结果相一致。最后，采集其中2头版纳微型猪近交系和1头巴马香猪的口腔黏膜样本进行免疫荧光检测，结果与血液PCR鉴定结果一致。结论通过采集胎儿细胞和活体猪的口腔黏膜样本进行PCR检测，可准确、高效地鉴定出猪的血型，为异种器官移植供体猪的血型筛选提供了一种简便方法。

关键词: 猪, 血型, PCR技术, 异种器官移植, 血液, 胎儿成纤维细胞, 口腔黏膜

Abstract:

Objective Xenotransplantation is an effective way to address the shortage of human organ donors, but it faces serious immune rejection reactions, including hyperacute rejection caused by blood type differences. Establishing a stable, convenient, and reliable method for pig blood type identification can quickly screen suitable donor pigs for xenograft research. Methods Banna miniature inbred pigs, Diannan small eared pigs, and Bama Xiang pigs were selected as the research objects. DNA was extracted from the blood, oral buccal mucosa, and fetal fibroblasts of the three strains of pigs using DNA extraction kits. The target fragment of the ABO homologous gene EAA intron 7 in pigs was amplified using PCR method. Blood agglutination reaction was used to detect hemolysis in pig anterior vena cava whole blood after adding anti A and B antibodies. Immunohistochemical method was used to detect the expression level of A antigen in pig heart, liver, spleen, lung, and kidney tissues. Immunofluorescence method was used to detect the expression level of A antigen in pig oral mucosa. By comparing the results of different methods for determining pig blood types, the stability and reliability of the PCR method were verified, and a convenient PCR based pig blood type identification method was established. Results Firstly, the blood PCR results of 69 inbred strains of Banna miniature pigs, 7 Diannan small eared pigs, and 34 Bama Xiang pigs showed 20 AO blood types, 66 AA blood types, and 24 O blood types. The PCR results of fetal fibroblasts from 47 Diannan small eared pigs showed that all 47 fetuses were O blood type. Among them, the oral mucosal PCR results of 8 gene edited cloned pigs were consistent with those of donor fetal fibroblasts, all of which were O blood type. The oral mucosal PCR results of 8 wild-type pigs (2 inbred lines of Banna miniature pigs, 4 Diannan small eared pigs, and 2 Bama Xiang pigs) were consistent with the blood PCR identification results. Then, 11 inbred lines of Banna miniature pigs, 4 Diannan small eared pigs, and 2 Bama Xiang pigs were randomly selected for blood agglutination reaction validation, and the results were consistent with the PCR identification results of both blood samples and oral mucosa samples. Moreover, immuno-histochemical analysis was performed on the heart, liver, lung, kidney, and spleen tissues of one Banna miniature pig inbred line and two Bama Xiang pigs, and the results were consistent with blood PCR identification and blood agglutination reaction results. Finally, oral mucosal samples were collected from 2 inbred strains of Banna miniature pigs and 1 Bama Xiang pig for immunofluorescence detection, and the results were consistent with the blood PCR identification results. Conclusion By collecting fetal cells and oral mucosal samples from live pigs for PCR detection, the blood type of pigs can be accurately and efficiently identified, providing a convenient method for blood type screening of xenograft donor pigs.

Key words: Pigs, Blood type, PCR technology, Xenotransplantation, Blood, Fetal fibroblasts, Oral mucosa

中图分类号:

R-332

王娇祥, 王艳, 胡珂, 徐凯祥, 魏太云, 角德灵, 赵恒, 赵红业, 魏红江. 猪血型PCR鉴定方法的建立[J]. 实验动物与比较医学, 2023, 43(6): 585-594.

Jiaoxiang WANG, Yan WANG, Ke HU, Kaixiang XU, Taiyun WEI, Deling JIAO, Heng ZHAO, Hongye ZHAO, HongJiang WEI. Establishment of PCR Identification Method for Pig Blood Type[J]. Laboratory Animal and Comparative Medicine, 2023, 43(6): 585-594.

图/表 7

图1 PCR方法鉴定猪血型的原理示意图（猪A和O血型EAA基因差异）

Figure 1 Schematic diagram of the PCR method for identifying pig blood types （differences in EAA gene between pig A and O blood types）

图2 不同品系猪血液血型PCR鉴定结果注：A，69头版纳微型猪近交系猪（BNP01～69）的血液PCR鉴定结果；B，7头滇南小耳猪（DNP01～7）的血液PCR鉴定结果；C，34头巴马香猪（BMP01～34）的血液PCR鉴定结果。M12000、M5000和M2000是DNA Marker。AOC、AAC和OOC分别指本实验室前期做过测序的AO、AA、OO血型标准对照样品。

Figure 2 PCR identification results of blood types in different strains of pigsNote：A， The blood PCR identification results of 69 Banna miniature inbred pigs （BNP01-69）； B， The blood PCR identification results of 7 Diannan small eared pigs （DNP01-7）； C， The blood PCR identification results of 34 Bama Xiang pigs （BMP01-34）. M12000， M5000 and M2000 are DNA Markers. AOC， AAC， and OOC respectively refer to the AO， AA， and OO blood group standard control samples that have been sequenced in our laboratory in the previous stage.

图3 滇南小耳猪胎儿成纤维细胞及其克隆猪口腔黏膜细胞的PCR鉴定血型结果注：A，17头野生型滇南小耳猪胎儿成纤维细胞（DNF01～17）的PCR鉴定结果；B，18头基因编辑滇南小耳猪胎儿成纤维细胞（DNF18～35）的PCR鉴定结果；C，12头基因编辑滇南小耳猪胎儿成纤维细胞（DNF36～47）的PCR鉴定结果；D，8头基因编辑克隆滇南小耳猪（DNP08～15）的口腔黏膜样品PCR鉴定结果；E，8头野生型猪（BNP11～12为2头版纳微型猪近交系；DNP01、03、04、06为4头滇南小耳猪；BMP31～32为2头巴马香猪）的口腔黏膜样品PCR鉴定结果。M2000是DNA Marker。AOC、AAC和OOC分别指本实验室前期做过测序的AO、AA、OO血型标准对照样品。

Figure 3 PCR identification of blood types in fetal fibroblasts and oral mucosal cells of Diannan cloned small eared pigsNote：A， PCR identification results of 17 wild-type Diannan small eared pig fetal fibroblasts （DNF1-17）； B， PCR identification results of 18 porcine gene-editing fetuses fibroblasts of Diannan small eared pigs （DNF18-35）； C， PCR identification results of 12 porcine gene-editing fetuses fibroblasts of Diannan small eared pigs （DNF36-47）；D， PCR identification results of oral mucosal samples from 8 gene-editing cloned Diannan small eared pigs （DNP08-15）； E， PCR identification results of oral mucosal samples from 8 wild- type pigs （BNP11-12， Two Banna miniature inbred pigs； DNP01，03，04 and 06， four Diannan small eared pigs； BMP31-32， two Bama Xiang pigs）. M2000 is the DNA Marker. AOC， AAC， and OOC respectively refer to the AO， AA， and OO blood group standard control samples that have been sequenced in our laboratory in the previous stage.

图4 血液凝集反应鉴定不同品系猪血型的结果注：A，11头版纳微型猪近交系（BNP02～08、10～13）的血液凝集反应；B，4头滇南小耳猪（DNP03～06）的血液凝集反应；C，2头巴马香猪（BMP31～32）的血液凝集反应。AA、AO、OO指血液PCR鉴定血型结果，与血液凝集反应结果一致。

Figure 4 Results of the blood agglutination reaction for identifying pig blood typesNote：A, Blood agglutination reaction of 11 Banna miniature inbred pigs (BNP02-08, 10-13); B, Blood agglutination reaction of Diannan small eared pigs (DNP03-06); C, Blood agglutination reaction of 2 Bama Xiang pigs (BMP31-32). AA, AO, and OO refer to the results of PCR identification of blood types, which were consistent with the results of blood agglutination reaction.

图5 各脏器免疫组织化学鉴定不同品系猪血型的结果（DAB染色，比例尺为100 μm）注：BNP07为版纳微型猪近交系，BMP02、24为巴马香猪。AA、AO、OO指血液PCR鉴定血型结果，与免疫组织化学法鉴定结果一致。

Figure 5 Results of immunohistochemical identification of pig blood types （DAB staining, all the bars are 100 μm）Note：BNP， Banna miniature inbred pigs； BMP， Bama Xiang pigs. AA， AO and OO refer to the results of blood type identification by PCR， which are consistent with the results of blood type identification by immunohistochemistry.

图6 口腔黏膜样品的免疫荧光鉴定不同品系猪血型的结果（比例尺为100 μm）注：BNP06、12为版纳微型猪近交系；BMP31为巴马香猪。FITC，异硫氰酸荧光素；DAPI，4'，6-二脒基-2-苯基吲哚。AA、AO、OO指血液PCR鉴定血型结果，与免疫荧光法鉴定血型结果一致。

Figure 6 Results of immunofluorescence identification of pig blood types in oral mucosal samples (all the bars are 100 μm)Note： BNP06， 12 are two Banna miniature inbred pigs； BMP31 is one Bama Xiang pig. FITC, Fluorescein isothiocyanate； DAPI， 4'，6-diamidine-2-phenylindole. AA， AO and OO refer to the results of blood PCR identification of blood types， which are consistent with the results of immunofluorescence identification of blood types.

表1 PCR、血液凝集反应、免疫组织化学和免疫荧光染色结果对比

Table 1 Comparison of PCR, hemagglutination test, immunohistochemistry and immunofluorescence staining results

序号Serial number	编号Number	血液 PCR Blood PCR	血液凝集反应 Hemagglutination test		免疫组织化学 Immunohistochemistry					颊黏膜PCR PCR of buccal mucosal samples	免疫荧光 Immunofluorescence
序号Serial number	编号Number	血液 PCR Blood PCR	抗A抗体Anti-A antibody	抗B抗体Anti-B antibody	心 Heart	肝 Liver	脾 Spleen	肺 Lung	肾 Kidney	颊黏膜PCR PCR of buccal mucosal samples	免疫荧光 Immunofluorescence
1	BNP02	AA	+	-
2	BNP03	AA	+	-
3	BNP04	AA	+	-
4	BNP05	AA	+	-
5	BNP06	AO	+	-							A
6	BNP07	AA	+	-	-	A	A	A	A
7	BNP08	AA	+	-
8	BNP09	AA	+	-
9	BNP10	AA	+	-
10	BNP11	AA	+	-						AA
11	BNP12	AA	+	-						AA	A
12	DNP03	AO	+	-						AO
13	DNP04	OO	-	-						OO
14	DNP05	OO	-	-
15	DNP06	AO	+	-						AO
16	BMP02	OO			-	-	-	-	-
17	BMP24	AO			-	A	-	A	A
18	BMP31	OO	-	-						OO	-
19	BMP32	OO	-	-						OO

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猪血型PCR鉴定方法的建立

Establishment of PCR Identification Method for Pig Blood Type

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