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Isolation of novel sequences targeting highly variable viral protein hemagglutinin.

Xu Z, Wu J, Feng F, Zhang X, Ma X, Tang M, Huang Y, Zhang Y, Cao Y, Cao W, He R, Gao Y, Liu Q - MethodsX (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: MOE Key Laboratory of Aquatic Product Safety, State Key Laboratory of Biocontrol, Biotechnology Research Center, The School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Colony PCR yielded 1550 bp products from E. coli transformants. M: molecular standard. Lanes 1–22 were PCR amplifications of 22 colonies.
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fig0015: Colony PCR yielded 1550 bp products from E. coli transformants. M: molecular standard. Lanes 1–22 were PCR amplifications of 22 colonies.

Mentions: The digested SK(−) vector and the HA assembly products were ligated with at an approximate molar ratio of 1:7 in the refrigerator (5 °C). The ligation mixtures were precipitated with ethanol and dissolved in 10 μl of sterile double distilled H2O or less, and electroporated to E. coli ElectroMax-DH10B strain (Invitrogen, Carlsbad, CA) [5]. Cells were plated onto LB/ampicillin plates. PCR was performed with primers 4 and 9 after microwave treatment of toothpick touched colonies [6], and 1550 bp PCR amplicons were visualized on agarose gel (Fig. 3). The positive rate was 54.5%, which was about 26-fold higher than that of the previous FMDV gene fragment libraries before the enrichment procedure [1]. The clones of E. coli DNA library carrying hemagglutinin gene fragment failed to grow, most likely resulted from stressful peptides expressed from the partial gene. Therefore the engineering of a full-length gene was advantageous, especially for genes from highly virulent viruses. Two of the putative positive clones were successfully sequenced and found to be novel. Bacteria were washed from the plates, and pooled, and treated as aforementioned. PCR amplicons were subsequently sequenced and shown to harbor multiple sequences (Fig. 4). The presence of virtually all the designed bases in the degenerated sites suggests that the library carried many clones.


Isolation of novel sequences targeting highly variable viral protein hemagglutinin.

Xu Z, Wu J, Feng F, Zhang X, Ma X, Tang M, Huang Y, Zhang Y, Cao Y, Cao W, He R, Gao Y, Liu Q - MethodsX (2015)

Colony PCR yielded 1550 bp products from E. coli transformants. M: molecular standard. Lanes 1–22 were PCR amplifications of 22 colonies.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4487337&req=5

fig0015: Colony PCR yielded 1550 bp products from E. coli transformants. M: molecular standard. Lanes 1–22 were PCR amplifications of 22 colonies.
Mentions: The digested SK(−) vector and the HA assembly products were ligated with at an approximate molar ratio of 1:7 in the refrigerator (5 °C). The ligation mixtures were precipitated with ethanol and dissolved in 10 μl of sterile double distilled H2O or less, and electroporated to E. coli ElectroMax-DH10B strain (Invitrogen, Carlsbad, CA) [5]. Cells were plated onto LB/ampicillin plates. PCR was performed with primers 4 and 9 after microwave treatment of toothpick touched colonies [6], and 1550 bp PCR amplicons were visualized on agarose gel (Fig. 3). The positive rate was 54.5%, which was about 26-fold higher than that of the previous FMDV gene fragment libraries before the enrichment procedure [1]. The clones of E. coli DNA library carrying hemagglutinin gene fragment failed to grow, most likely resulted from stressful peptides expressed from the partial gene. Therefore the engineering of a full-length gene was advantageous, especially for genes from highly virulent viruses. Two of the putative positive clones were successfully sequenced and found to be novel. Bacteria were washed from the plates, and pooled, and treated as aforementioned. PCR amplicons were subsequently sequenced and shown to harbor multiple sequences (Fig. 4). The presence of virtually all the designed bases in the degenerated sites suggests that the library carried many clones.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: MOE Key Laboratory of Aquatic Product Safety, State Key Laboratory of Biocontrol, Biotechnology Research Center, The School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus