Isolation of novel sequences targeting highly variable viral protein hemagglutinin
2015
Xu, Zhiwu | Wu, Jieyu | Feng, Fan | Zhang, Xiaojiao | Ma, Xiaoqian | Tang, Man | Huang, Yan | Zhang, Ying | Cao, Yongchang | Cao, Weiguo | He, Ran | Gao, Ye | Liu, Qiuyun
Rapid evolution is a hallmark of the viral kingdom and a major concern for developing universal vaccines. The isolation of substantial numbers of viral sequence variants at highly variable viral protein domains remains a major challenge. We previously developed a combinatorial method for the isolation of novel sequences to cope with rapid viral variations at the G-H loop of Foot and Mouth Disease virus VP1 protein [1]. Here we present a modification of that method in its application in the randomization of the hemagglutinin gene from a H5N2 virus, namely:•removal of potentially stressful region which harbored a stretch of basic amino acids to increase the success rates of gene cloning, and to streamline the process of future engineering of novel viral variants.•clustered randomization in a full-length gene, as the positive rate for partial gene fragment libraries was extremely low before enrichment in the previous FMDV studies.•the use of fusion partner was avoided, which was used previously for protein expression, stabilization of clones and reduction of stresses on host cells.•the use of Poisson distribution is proposed to approximate sequencing output to achieve cost effectiveness.
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