摘要
利用PCR方法在hCGβcDNA 3’端产生一个Bam HI酶切位点,将此cDNA克隆入pBS HindⅢ和Bam HI位点之间。用T3、T7引物从正反两个方向同时测序,确证其序列正确性。将C3d3 cDNA从pSG5·PSG5·C3d3·YL·C3d3·YL切出与hCGβ串联构建成质粒pBS-hCGβ-C3d3。然后构建表达载体pVL1393-hCGβ-C3d3,并与线性化核型多角体杆状病毒(AcNPV)基因组DNA共转染昆虫细胞Sf9,以噬斑法筛选出重组病毒AcNPV-hCGβ-C3d3。以重组病毒AcNPV-hCGp-C3d3转染Sf9细胞,收集的条件培液经检测,确定其具有远远高于hCGβ-oLHα的结合活性。经hCGβ单抗亲和层析柱分离纯化后,以SDSPAGE和Western blot均证实了表达产物的存在,分子量与预期的hCGβ-C3d3一致,并具有hCGβ的免疫学活性。
In view of the strong immunity-enhancing function of HEL-C3d3 designed by Dr. Paul W. Dempsey, we made our efforts to produce a similar recombinant protein of hCGβ. With poly-merase chain reaction, we introduced a Bam HI restriction site into the 3' terminal of hCGβ cD-NA. The new cDNA and its terminal's correctness has been confirmed by sequencing. Then we have it covalently attached to the C3d3 cDNA at the pre-designed Bam HI/Bgl II site.
Having the chimeric DNA correctly cloned into the protein nuclear polyhedrosis virus (AcN-PV) expression vector pVL1393, we constructed the expression vector pVL1393-(hCGβ-C3d3). The insect cells were co-transfected with the ex-
pression vector and linearized nuclear polyhedrosis virus DNA, and recombinant viruses AcNPV-(hCGβ-C3d3) were screened out. Through anti-hCGβ immunoaffnity chromatography, the recombinant hCGβ-C3d3 chimera polypeptide was purified from culture supernatant of insect cells infected by the recombinant viruses. In RIA test, the expressed product competitively inhibits the binding of 125I-hCGβ to hCGβ-antibody. On SDS-PAGE and Western blot, the recombinant peptide hCGβ-C3d3 obviously appears to be with a molecular weight of 116KD. Therefore, we arrive at a conclusion that it has a normal immuno-genic ability.
出处
《实验生物学报》
CSCD
1999年第1期31-37,共7页
Acta Biologiae Experimentalis Sinica
基金
国家自然科学基金
国家科委"九五"攻关资助项目
关键词
HCG
Β亚基
补体
单链多肽
生物合成
hCGβ-C3d3. Expression vector. Recombinant virus. Insect cell (Sf9). Expression
