山羊痘病毒毒力因子KLP1的表达及多克隆抗体的制备
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摘要
为了研究羊痘病毒KLP1的特性及制备其抗体,研究通过PCR扩增了山羊痘病毒毒力因子KLP1的两个结构域KLP1-1、KLP1-2,并构建了重组表达载体pET42b-KLP1-1和pET42b-KLP1-2,将重组载体转化到大肠杆菌BL21中经IPTG诱导表达,表达产物应用SDS-PAGE和Western-blot检测,表达蛋白经镍柱纯化或KCl染色切胶纯化,经弗氏佐剂乳化后免疫家兔,制备的抗体用Western-blot和ELISA检测评价。结果表明:克隆得到的KLP1-1基因的大小为762 bp,表达约为30 ku的蛋白,主要在上清液中表达,镍柱纯化蛋白浓度为3 mg/mL;KLP1-2的基因大小为927 bp,表达约为35 ku的蛋白,以包涵体形式表达在沉淀中,切胶纯化的蛋白浓度在1~2 mg/mL之间。免疫家兔后收集血清,经Western-blot检测可见明显的特异条带,ELISA检测的抗体效价分别为1∶512和1∶256。
The aim of the present study was to identify the characteristics of KLP1 and prepare the antibodies of KLP1. Two domains( KLP1-1 and KLP1-2) of Goat poxvirus virulence factor KLP1 were amplified by PCR and its prokaryotic expression vectors( pET42 b-KLP1-1 and pET42 b-KLP1-2) were constructed,respectively. The vectors were transformed into Escherichia coli BL21. Expression of the proteins were induced by IPTG. The proteins were identified by SDS-PAGE and Western-blot. The expressed protein was purified by Ni-NTA resin or gel slices after KCl staining. The rabbits were immunized with the purified proteins emulsified with Freund's adjuvant. The rabbit anti-sera was detected by Western-blot and ELISA. The results showed that the KLP1-1 gene was 762 bp and the expressed protein size was about 30 ku in supernatant which could be purified by Ni column. The concentration of the purified protein was 3 mg/mL. The KLP1-2 was 927 bp and the expressed as inclusion body. The protein size was about 35 ku in sediment which could be purified by gel slices after KCl staining. The concentration of the purified protein was 1-2 mg/mL. The anti-sera was collected after immunization of rabbits. The antibodies of KLP1-1 and KLP1-2 protein had been detected by Western-blot,and its titers of serum were 1 ∶512 and 1 ∶256 by ELISA detection,respectively.
The aim of the present study was to identify the characteristics of KLP1 and prepare the antibodies of KLP1. Two domains( KLP1-1 and KLP1-2) of Goat poxvirus virulence factor KLP1 were amplified by PCR and its prokaryotic expression vectors( pET42 b-KLP1-1 and pET42 b-KLP1-2) were constructed,respectively. The vectors were transformed into Escherichia coli BL21. Expression of the proteins were induced by IPTG. The proteins were identified by SDS-PAGE and Western-blot. The expressed protein was purified by Ni-NTA resin or gel slices after KCl staining. The rabbits were immunized with the purified proteins emulsified with Freund's adjuvant. The rabbit anti-sera was detected by Western-blot and ELISA. The results showed that the KLP1-1 gene was 762 bp and the expressed protein size was about 30 ku in supernatant which could be purified by Ni column. The concentration of the purified protein was 3 mg/mL. The KLP1-2 was 927 bp and the expressed as inclusion body. The protein size was about 35 ku in sediment which could be purified by gel slices after KCl staining. The concentration of the purified protein was 1-2 mg/mL. The anti-sera was collected after immunization of rabbits. The antibodies of KLP1-1 and KLP1-2 protein had been detected by Western-blot,and its titers of serum were 1 ∶512 and 1 ∶256 by ELISA detection,respectively.
引文
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[6]PIRES de MIRANDA M,READING P C,SMITH G L,et al.The vaccinia virus kelch-like protein C2L affects calcium-independent adhesion to the extracellμLar matrix and inflammation in a murine intradermal model[J].J Gen Virol,2003,84(Pt9):2459-2471.
[7]BALINSKY C A,DELHON G,AFONSO C L,et al.Sheeppox virus kelch-like gene SPPV-019 affects virus virulence[J].J Virol,2007,81(20):11392-11401.
[8]LI Y W,JIAO H H,HU J J,et al.Identification of four goatpox virus outbreaks in xinjiang of China in 2010[C]//IEEE.Collected papers of international conference on human health and biomedical engineering in 2011.Jilin:[s.n.],2011:1003-1006.
[2]TULMAN E R,AFONSO C L,LU Z,et al.The genomes of sheeppox and goatpox viruses[J].J Virol,2002,76(12):6054-6061.
[3]ADAMS J C,KELSO R,COOLEY L,et al.The kelch repeat superfamily of proteins:propellers of cell function[J].Trends Cell Biol,2000,10(1):17-24.
[4]AROMOLARAN K A,BENZOW K A,KOOB M D,et al.The Kelch-like protein 1 modulates P/Q-type calcium current density[J].Neuroscience,2007,145(3):841-850.
[5]SHCHELKUNOV S,TOTMENIN A,KOLOSOVA I,et al.Speciesspecific dif-ferences in organization of orthopoxviruskelch-like proteins[J].Virus Genes,2002,24(2):157-162.
[6]PIRES de MIRANDA M,READING P C,SMITH G L,et al.The vaccinia virus kelch-like protein C2L affects calcium-independent adhesion to the extracellμLar matrix and inflammation in a murine intradermal model[J].J Gen Virol,2003,84(Pt9):2459-2471.
[7]BALINSKY C A,DELHON G,AFONSO C L,et al.Sheeppox virus kelch-like gene SPPV-019 affects virus virulence[J].J Virol,2007,81(20):11392-11401.
[8]LI Y W,JIAO H H,HU J J,et al.Identification of four goatpox virus outbreaks in xinjiang of China in 2010[C]//IEEE.Collected papers of international conference on human health and biomedical engineering in 2011.Jilin:[s.n.],2011:1003-1006.