Analysis of the characteristics of porcine antibody repertoire by high-throughput sequencing method

doi:10.3785/j.issn.1008-9209.2020.10.202

Journal of Zhejiang University (Agriculture and Life Sciences)

2021, Vol. 47

Issue (5): 660-666 DOI: 10.3785/j.issn.1008-9209.2020.10.202

Animal sciences & veterinary medicine

Analysis of the characteristics of porcine antibody repertoire by high-throughput sequencing method

Chunmiao JI^1,²(

),Bin WANG¹,Pan QIN¹,Yaowei HUANG¹(

)

^1.Institute of Preventive Veterinary Medicine, College of Animal Sciences, Zhejiang University, Hangzhou 310058, China
^2.Zhaoqing Branch Center of Guangdong Laboratory for Lingnan Modern Agricultural Science and Technology, Zhaoqing 526238, Guangdong, China

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Abstract

The adaptive immune system can protect the body from various pathogens by producing a variety of antibodies. The antibody repertoire is so vast that the traditional low-throughput sequencing method cannot completely sequence it. In this study, the library of porcine B cell receptor (BCR) heavy chain was amplified from porcine peripheral blood mononuclear cell (PBMC) by multiplex polymerase chain reaction (PCR). The characteristics of porcine antibody repertoire were analyzed by next-generation sequencing. The results showed that several IGHV, IGHD and IGHJ genes were preferentially used by three-week-old piglets under normal conditions, and IGHV1-4 and IGHV1S2 were the two most frequently used variable (V) genes. The total frequencies of the use of eight IGHV genes accounted for more than 80%. In addition, the length of heavy chain complementarity determining region 3 (HCDR3) was a normal distribution with the average length of 15.8 amino acids. This study successfully constructs a high-throughput sequencing method suitable for porcine antibody repertoire, which provides an effective approach for further understanding the specific composition of it in physiological and pathological states.

Key words： pig antibody repertoire composition high-throughput sequencing method

Received: 20 October 2020 Published: 27 October 2021

CLC:

S 855.3

Corresponding Authors: Yaowei HUANG E-mail: jichunmiaomiao@163.com;yhuang@zju.edu.cn

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Cite this article:

Chunmiao JI,Bin WANG,Pan QIN,Yaowei HUANG. Analysis of the characteristics of porcine antibody repertoire by high-throughput sequencing method. Journal of Zhejiang University (Agriculture and Life Sciences), 2021, 47(5): 660-666.

URL:

http://www.zjujournals.com/agr/10.3785/j.issn.1008-9209.2020.10.202 OR http://www.zjujournals.com/agr/Y2021/V47/I5/660

利用高通量测序技术分析猪抗体库的组成特征

宿主适应性免疫系统通过产生种类多样的抗体保护其免受病原侵害，而传统的低通量测序方法无法完成对宿主抗体库的测序。本研究通过多重聚合酶链反应（polymerase chain reaction, PCR）从猪外周血单个核细胞（peripheral blood mononuclear cell, PBMC）中扩增获得猪B细胞受体（B cell receptor, BCR）重链文库，经二代测序建立并分析健康猪抗体库的组成特征。结果表明：3周龄仔猪在正常情况下对抗体库IGHV、IGHD、IGHJ基因使用具有偏好性，其中IGHV1-4和IGHV1S2是使用最频繁的2种V（variable）基因，有8种IGHV基因的使用频率之和超过80%，另外发现重链互补决定区3（heavy chain complementarity determining region 3, HCDR3）的长度呈正态分布，平均长度约为15.8个氨基酸。本研究成功建立了适用于猪抗体库的高通量测序方法，可为未来进一步深入理解不同状态（生理、病理）猪抗体库的特异性组成提供有效的研究方法。

关键词： 猪, 抗体库, 组成, 高通量测序, 方法

Fig. 1 High-throughput sequencing method for the porcine antibody repertoireA. Flow chart of high-throughput sequencing of the porcine antibody repertoire; B. Scheme for multiplex PCR amplification of porcine BCR heavy chain library.

Table 1 Primers used for multiplex PCR amplification

Table 2 Summary of sequencing reads alignment

Fig. 2 Usage of IGHV gene in the porcine antibody repertoire

Fig. 3 Usage of IGHD gene in the porcine antibody repertoire

Fig. 4 Usage of IGHJ gene in the porcine antibody repertoire

Fig. 5 Distribution of HCDR3 with different lengths in four pigletsHCDR3: Heavy chain complementarity determining region 3.


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