Codon Optimization and Expression of Bovine Pregnancy-related Glycoprotein-16 Gene
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摘要:
目的 对bPAG16基因进行优化和合成，构建proEM-bPAG16重组表达载体，将重组质粒转染至HEK293细胞中，获得bPAG16重组蛋白，为家畜早期妊娠诊断技术的研发提供技术支撑。 方法 通过生物学技术对bPAG16基因密码子进行优化和人工合成，经T4 DNA连接酶将bPAG16基因插入到proEM载体中，构建重组载体proEM-bPAG16，将其转染至HEK293细胞中进行瞬时表达，重组蛋白经Ni-IDA亲和柱纯化后，采用SDS-PAGE、Western Blot检测其表达效果。 结果 优化后的bPAG16基因密码子适用指数（CAI）由原来的0.77提高到0.96，GC含量由48%提高到58%。重组载体proEM-bPAG16双酶切后分别获得1 179 bp和4 369 bp的2条片段，与预期值一致；重组质粒测序结果显示，其核苷酸序列与优化后基因完全一致，氨基酸未发生突变；SDS-PAGE 和 Western Blot 鉴定结果显示，获得重组蛋白bPAG16（rbPAG16）相对分子质量约48 kDa，经Ni-IDA亲和层析纯化后，其纯度达到90%以上。 结论 通过密码子优化策略及重组蛋白技术高效表达了rbPAG16，为奶牛早孕快速检测技术的研发奠定了基础。 Abstract:Objective The bPAG16 gene related to bovine pregnancy was codon-optimized and synthesized to construct recombinant vector expressed in HEK293 cells for the development of a diagnostic tool on pregnancy of dairy cows. Methods Bioinformatics were applied to codon-optimize and synthesize the bPAG16 gene followed by connecting it to the proEM vector by T4 DNA ligase. The resulting proEM-bPAG16 recombinant vector was transfected into HEK293 cells. The expressed gene was purified by Ni2+ affinity chromatography and verified by SDS-PAGE and western blotting. Result With the optimization, the codon adaptation index (CAI) of the bPAG16 gene rose from 0.77 to 0.96 and the GC content from 48% to 58%. After enzymatic digestion, fragments of approximately 1 179 bp and 4 369 bp were obtained from the proEM-bPAG16 expression vector as expected. The nucleotide sequence of the inserted bPAG16 in the recombinant plasmid was same as that of the optimized gene and absent of any mutated amino acids. SDS-PAGE and western blotting showed that the 48kDa recombinant bPAG16 fusion protein was correctly expressed in the HEK293 cells with a purity greater than 90%. Conclusion The successfully executed codon optimization and synthesis of bPAG16 gene provided the base material for further study on early diagnosis of pregnancy in dairy cows. -
图 1 bPAG16基因优化前后核苷酸、氨基酸对比结果
注：下划线为优化后基因与原始基因的差异碱基位点；粗体是起始密码子和终止密码子；方框内字体为Eco RI、Hind III 酶切位点；5′端斜体为保护碱基序列，3′端斜体为His标签序列。
Figure 1. Nucleotide alignment of original and optimized bPAG16 genes
Note: Underlined nucleotide sites differ between original and codon-optimized bPAG16 gene. Boldface letters indicate initial and ending codons. Boxed letters are Eco RI and Hind III enzyme cleavage sites. Italics on 5' end are kozak sequence, and on 3' end His label sequence.
图 2 bPAG16 基因优化前后的密码子适用指数对比
注：A：优化前CAI指数，B：优化后CAI指数。
Figure 2. CAIs of bPAG16 gene before and after codon optimization
Note: A: CAI of original bPAG16 gene; B: CAI of codon-optimized bPAG16 gene.
图 3 bPAG16
基因优化前后的GC含量对比 注：A：优化前GC含量，B：优化后GC含量。
Figure 3. GC contents of bPAG16 gene before and after codon optimization
Note: A: GC content of original bPAG16 gene; B: GC content of codon-optimized bPAG16 gene.
图 4 优化bPAG16基因的PCR扩增产物
注：M：DNA Marker；1～2：PCR产物。
Figure 4. Electropherogram of PCR products of codon-optimized bPAG16
gene Note: Lane M: DNA marker; Lanes 1&2: PCR products.
图 5 阳性克隆（A）及重组质粒双酶切（B）鉴定结果
注：M：DNA Marker；A：1-8：阳性克隆；B：1：重组质粒，2：双酶切片段。
Figure 5. Identification of cloning products (A) and recombinant plasmid (B)
Note: Lane M: DNA marker; A: Lanes 1-8: positive clones; B: Lane 1: intact recombinant plasmids, Lanes 2: digested recombinant plasmids.
图 6 SDS-PAGE分析重组蛋白bPAG16
注：M：蛋白 Marker，1：上清液，2：流出液，3～5：50 mmol·L−1 咪唑洗脱组分，6～8：500 mmol·L−1咪唑洗脱组分。
Figure 6. SDS-PAGE analysis on rbPAG16
Note: Lane M: protein marker; Lane 1: supernatant; Lane 2: flow-through; Lanes 3-5: eluent containing 50 mmol·L−1 imidazole; Lanes 6-8: eluent containing 500 mmol·L−1 imidazole.
图 7 重组蛋白bPAG16的Western blot鉴定
注：M：蛋白Marker，1：重组蛋白bPAG16（0.5 μg）。
Figure 7. Western blotting analysis on rbPAG16
Note: Lane M: protein marker; Lane 1: rbPAG4.
表 1 bPAG16基因密码子偏好性分析
Table 1. Codon usage of bPAG16 gene in Ampullaria crossean
氨基酸 Amino acid 密码子 Condon 相对使用度 RSCU 氨基酸 Amino acid 密码子 Condon 相对使用度 RSCU 异亮氨酸 IIe ATA 0.32 组氨酸 His CAT 1.11* ATT 1.29* CAC 0.89 ATC 1.39* 缬氨酸 Val GTC 0.67 酪氨酸 Tyr TAC 1.57* GTG 1.87* TAT 0.43 GTT 0.93 GTA 0.53 赖氨酸 Lys AAA 0.78 天冬氨酸 Asp GAT 0.80 AAG 1.22* GAC 1.20* 脯氨 Pro CCT 1.05* 谷氨酰胺 Gln CAA 1.14* CCG 0.42 CAG 0.86 CCC 1.05* CCA 1.47* 亮氨酸 Leu CTA 0.71 谷氨酸 Glu GAG 1.60* CTC 1.24* GAA 0.40 TTG 1.06* CTG 2.12** CTT 0.71 TTA 0.18 精氨酸 Arg AGG 1.20* 色氨酸 Trp TGG 1.00 AGA 2.80** CGT 0.20 CGG 0.80* CGA 0.60 CGC 0.40 苏氨酸 Thr ACC 1.67* 半胱氨酸 Cys TGC 0.75 ACG 0.33 TGT 1.25* ACT 1.00 ACA 1.00 甲硫氨酸 Met ATG 1.0 丙氨酸 Ala GCC 2.12** GCG 0.71 GCA 0.71 GCT 0.47 丝氨酸 Ser AGT 0.77 苯丙氨酸 Phe TTC 1.05* AGC 0.77* TTT 0.95 TCC 0.77 TCT 2.52** TCA 1.16* TCG 0.00 甘氨酸 Gly GGA 0.88 天冬氨酰 Asn AAC 1.13* GGC 1.25* AAT 0.94 GGT 1.00 GGG 0.88 注：*、**：高表达偏好性密码子。
Note: *, **indicate codon preference.
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