| Now little is known about the effect of porcine LH-β gene on the reproduction and immunity. And there still lack the systematic explorations on the transgenetic expression of LH-β gene in vivo and in vitro, and the expression effect of porcine LH-β gene in vivo on the reproduction and immune system of animals. Our experiment is conducted to clone porcine LH-β gene from hypophysis of the special Meishan pig and systematically study its expressing effect in vivo and in vitro on the reproduction and immune system. It is expected to develop a novel way to upregulate reproduction physiological responses of animal to increase the pregnancy and birth of young animals.Porcine LH-β cDNA from hypophysis was synthesized by RT-PCR from mRNA extracted from Meishan pig. The porcine LH-p cDNA was then subcloned into pMDIS-Tvector and named as pUPLH- β . The sequence of cloned porcine LH-β cDNA was determined with M13/pUC sequence primer using BigDye Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer, USA) by ABI PRISM 377 DNA sequencer. The length of the LH-βgene was 417 bp, encoding a peptide of 139 amino acids whose molecular weight is 14.7 KDa and pi is 7.80. The signal sequence of peptides is from 1 to 30 bp and the mature peptides from 31 to 448 bp. By blasting the homologous sequences in GenBank databases, the sequence of porcine LH-β cDNA from lymphocyte is 98% homologus compared with the previously cloned porcine LH-β cDNA.The PLH-β cDNA fragment was inserted into pGEX-1λT plasmid to construct the expression plasmid pGPLH- β, the recombinant plasmid was digested by BamH I and EcoRI to identify whether the PLH-β cDNA fragment was inserted into the plasmid in correct orientation, the pGPLH- β was transformed into E. coli DH5a competent cells. The mRNA and protein expression were assayed by RT-PCR and SDS-PAGE. The results were found that the specific 417bp DNA bases of LH- β was detected by RT-PCR in the recombinant bacteria and a new protein band was found in SDS-PAGE with molecular mass of about 41 KDa which is consisted of a 14 KDa protein deduced from the LH- β gene sequence and GST (26 KDa). These indicated that the porcine LH- β gene was correctly transcribed and translated in the recombinant bacteria. The fusion protein was recovered from polyacrylamid gel and used to immunize the rabbit, and the titer of rabbit anti-porcine LH- β serum from immunized rabbit was 1: 51,200.The pUPLH- β was digested with BamH Dand Bglll, and then the insert was recovered from agarose gel using Agarose Gel DNA Extraction Kit. VR1020 was used to construct the eukaryotic expression plasmid, anddigested with BamHDand Bglll, andthen the digested LH- β fragment was ligated with the digested VR1020. The recombinant plasmid was named VPLH- β . VPLH- β was transformed into E. coli DH5a competent cells. The recombinant plasmid were extracted and digested with BamH I and Bglll to identify the correct insert orientation. The ability of VPLH- P to express full-length porcine LH- β in vitro was confirmed by RT-PCR in COS7 cells that were transiently transfected with VPLH- β in the entrappment of Lipofectamine.78 BAL B/c female mice at the age of 2 weeks were divided into 6 groups and were vaccinated intramuscularly in the left and right quardriceps respectively with l00ug VPLH-β entrapped with cationic liposome, with 100ug VPLH-P, 100ug recombinant PLH-β protein, with 10U PMSG and 20U HCG; Saline solution was used as control treatment. The blood of mice were collected from tail vein on 7, 14, 21, 28 days after inoculation to determine the dynamic changes of immunity and the content of LH- β and FSH in the sera. The experimental results indicated that the number of immune cells significantly increased respectively in the mice inoculated with VPLH- β and PLH-β, compared with those of the control; the IgG content mounted remarkably in the sera from the VPLH- β and PLH-β inoculated mice; the amount of LH and FSH were significantly raised in the VPLH-β plasmid and P...
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