[Study on the preparing of polyclonal antibodies against plant-selected maker gene hpt expression protein by DNA immunization].
OBJECTIVE
To prepare the polyclonal antibodies (pAbs) against HPT, a kind of plant-selected maker gene encoding protein, by DNA immunization technique and explore the influencing factors for this gene immunization.


METHODS
The coding sequence of hpt was cloned into eukaryotic expression vector pCDNA3. The sequence of the plasmid pCDNA3-HPT was demonstrated by restricting enzyme digestion analysis and DNA sequencing. The sequence-correct recombinant plasmids were purified and used to immunize BALB/c mice. The titer and specificity of antisera were detected by ELISA and Western blot, respecitively.


RESULTS
No pAb against HPT was detected following three immunization with hpt genes. Then mice were divided into three groups when the forth booster immunization was carried out: 1st group (immunization with the endotoxin-free recombinant plasmids), 2nd group (immunization with (His)(6)-HPT fusion protein expressed in E.coli) and 3rd group (immunization with the same plasmids as before mentioned). As a result, the pAb titer of the 1st group mice increased to 1:200, and the that of 2nd group was up to 1:2 000. Yet the 3rd group detected no anti-HPT antibody. Western blot analysis had proved that antisera of the first two groups could produce specific binding reaction to the purified GST-HPT, (His)(6)-HPT protein and their expressed product (bacterial protein).


CONCLUSIONS
We have got successfully the specific pAb against HPT by DNA immunization, but its titer is yet unsatisfactory, inferring that the character of the hpt gene self and its expression level may play an important role. In order to raise level of serum antibody prepared by DNA immunization, the farther study on various influencing factors still need to be performed.
Yang
Li-Chen
LC
Key Laboratory of Trance Element Nutrition of the Ministry of National Health, Institute of Nutrition and Food Safety, Chinese Center for Diseases Control and Prevention, Beijing 100050, China.
Zhu
Zhen
Z
Yang
Xiao-Guang
XG
D000818:Animals; D000906:Antibodies / Q000276:immunology; D004247:DNA / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007114:Immunization; D019444:Vaccines, DNA / Q000276:immunology
D000906:Antibodies; D019444:Vaccines, DNA; D004247:DNA