Part I: Construction Of Recombinant Viral Vector VAc EGT, Its Application And Infection In Silkworm Part II: Production And Application Of Rabbit Anti-synthetic Spider Dragline Protein Polyclonal Antibody

The silkworm Bombyx mori, is a silk-spinning domestic lepidopteran insect with both economic and scientific importance. Silkworm has been wildly regarded as the model organism of lepidopteran insects because of its relative better research background and convenient culture conditions among the insects. However, when compared with other model organisms such as the fruit fly, research on the silkworm still lagged behind. One of the important reasons is the shortage of the research tools for functional study of silkworm genes, which has became a limitation for further study. Among the tools waiting for improvement or development, a gene vector system for in vivo delivery and transient expression of foreign genes is needed. The front half part of the first part showed the feasibility of the application of recombinant baculovirus vAc?EGT as a foreign gene deliver and expression vector for the silkworm in vivo. Using enhanced green fluorescent protein (EGFP) as the reporter, work in this part proved in vivo gene transfer and high level transient expression of foreign gene carried by vAc?EGT can be achieved in various kinds of tissue cells, when budded virions (BV) of vAc?EGT were directly injected into haemocoel of newly ecdysed 5th instar larvae of the silkworm strain 54A. Recombinant bacolovirus vAc?EGT was constructed by deletion of the ecdysteroid UDP-glucosyltransferase (EGT) gene from the genome of Autographa californica nuclear polyhedrosis virus (AcNPV). EGT gene is a viral nonessential early gene, whose encoded product can inactivate the insect ecdysteroids using a sugar conjugation reaction, which results in prolonged larval instar and excess production of virus in hosts. High level expression of the EGT gene after injection of high titer AcNPV into silkworm larvae has also been demonstrated to alter the moulting and metamorphosis, and even cause the death of the silkworm, which is the virus'non-target insect. And the moulting or metamorphosis of silkworms will not be altered, if the insects received injection of EGT-null AcNPV. EGFP reporter delivered by EGT-null AcNPV vector vAc?EGT and controlled by different silkworm promoters were expressed in silkworm in vivo. When controlled by silkworm Actin 3 (A3) promoter, EGFP was expressed ubiquitously in various tissues; when controlled by silkworm fibroin light chain promoter, EGFP was specifically expressed in silk gland; when controlled by silkworm fibroin heavy chain promoter with the coding region of secretion signal peptide, EGFP was expressed and secreted into the cavity of silk gland. The results indicated that foreign genes delivered by vAc?EGT could be expressed in silkworm in vivo and vAc?EGT could serve in functional study of certain questions in vivo such as the mechanisms underlying the tissue specific transcription, expression and silk protein secretion in silk gland. EGT-null AcNPV vAc?EGT shows no influence over the instar duration of silkworm larvae. To our surprise, vAc?EGT or AcNPV with wild type EGT gene could infect and cause the death of silkworm larvae of the strain 54A, when foreign genes were delivered and efficiently expressed in the insect larvae in vivo. However, the general belief is either the intrahemocoelical infection, or the oral infection of AcNPV to silkworm will fail. The second half of the first part reported the novel phenomenon that AcNPV could infect partial strains of silkworm and characteristics of the infection, using the infection of silkworms by vAc?EGT carrying EGFP reporter as the model. The host genes involved in susceptibility of silkworm to AcNPV were also investigated by genetic cross experiments. Totally 35 kinds of strains randomly picked from Chinese strains, European strains and Japanese strains were assayed for their susceptibility to vAc?EGT-EGFP by intrahemocoelical infection. We found 16 strains including some Chinese strains, European strains and Japanese strains could be infected by vAc?EGT-EGFP, indicating the susceptibility of silkworm to AcNPV is a common phenomenon. When intrahemocoelically infected, viral titer in larvalhaemolymph of permissive strains rapidly increased and maintained between 107~108 PFU / ml, and viral DNA in tissues of larvae increased as well, which strongly suggested the numerous replication and propagation of AcNPV in silkworm. High level expression of EGFP reporter controlled by a very late viral promoter, the polh promoter, in tissues including hemocytes, fat bodies and silk glands also demonstrated the severe infection of AcNPV. To briefly address the genetic trait of silkworm anti-AcNPV genes, we did a series of genetic cross experiments to assay whether vAc?EGT-EGFP could infect the hybrid offspring of a permissive strain and a nonpermissive strain, Haoyue×Qingsong, the inbred offspring of Haoyue×Qingsong, and backcross of Haoyue×Qingsong to Haoyue or to Qingsong. The results strongly suggested that silkworm's resistance to intrahemocoelical infection of AcNPV was determined by only one antiviral gene or one set of genetically linked antiviral genes, whose function is dominant. This gene (genes) exists on some euchromosome but not sex chromosome of the nonpermissive silkworm strains, and is absent or inactivated in AcNPV permissive strans. Identification and further study of the antiviral gene would provide insights into the molecular basis of viral host range restriction and how organisms evolve inhibitor genes or certain defence mechanisms to prevent the viral infection. Work presented in the second part was production and application of rabbit anti-synthetic spider dragline polyclonal antibody. A synthetic spider dragline gene s600 was cloned into fusion protein expression vector pGEX-KG and expressed in Escherichia coli. Protein S600 was purified and rabbit antiserum was prepared. Amino acid composition analysis confirmed the right expression of S600. Western blot analysis revealed that anti-S600 antiserum can react with natural spider silk, so the synthetic dragline protein we designed shares similar immunological characteristics with the natural spider silk. We prepared the anti-S600 antiserum for screen and identification of the potential transgenic silkworms expressing artificial spider dragline in the silk gland and cocoons, as well as semi-quantification of dragline protein expressed in cocoons. We mixed the purified S600 protein with natural cocoon protein in certain proportion to mimic the expression of artificial spider dragline in cocoons of transgenic silkworms. ELISA results indicated that when the weight ratio of S600 to cocoon protein is between 0.06:10 to 0.12:10, that...